Methods for enhancement of protective immune responses

ABSTRACT

Methods for eliciting or enhancing immune responses to antigens, including tumor antigens, and/or DNA vaccines are provided. The methods employ polypeptides or nucleic acid compositions that contain at least a biologically active portion of a  Leishmania braziliensis  or  Leishmania major  homologue of the eukaryotic initiation factor 4A, or a variant thereof. Such polypeptides and compositions are useful for enhancing or eliciting a patient&#39;s cellular and/or humoral immune response, for instance within methods for treating tumors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 08/989,370filed Dec. 12, 1997, issued as U.S. Pat. No. 6,013,268 on Jan. 11, 2000,which is a continuation-in-part of U.S. application Ser. No. 08/634,642,filed Apr. 18, 1996, issued as U.S. Pat. No. 5,879,687 on Mar. 9, 1999,which is a continuation-in-part of U.S. application Ser. No. 08/607,509filed Feb. 23, 1996, issued as U.S. Pat. No. 5,876,735 on Mar. 2, 1999,which is a continuation-in-part of U.S. application Ser. No. 08/488,386filed Jun. 6, 1995, now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 08/454,036 filed May 30, 1995, issued as U.S.Pat. No. 5,876,966 on Mar. 2, 1999, which is a continuation-in-part ofU.S. application Ser. No. 08/232,534 filed Apr. 22, 1994, now abandoned.

TECHNICAL FIELD

The present invention relates generally to compounds and methods forenhancing immune responses in patients, as well as in isolated cells andcell cultures. The invention is more particularly related to compoundscomprising all or a portion of a Leishmania antigen that is a homologueof the eukaryotic initiation factor 4A (eIF4A), and to the use of suchcompounds in vaccines for stimulating immune responses.

BACKGROUND OF THE INVENTION

Vaccines commonly induce immunity to an infection or a disease bygenerating an immune response in a patient to a specific antigenassociated with the infection or disease. Modern techniques for theidentification and use of appropriate antigens have the potential tolead to the testing and development of a large number of vaccinesspecific for common infections (including bacterial, viral and protozoaninfections), as well as diseases such as cancer.

However, in many cases, purified antigens are weak immunogens, i.e., theimmune response generated by a specific antigen, while directed againstthe desired target, is not of a sufficient magnitude to confer immunity.In such cases, an immunomodulating agent, such as an adjuvant orimmunostimulant, must be employed to enhance the immune response.Adjuvants are substances that enhance a specific immune response to anantigen when injected with the antigen or at the same site as theantigen. Such substances function by a variety of mechanisms, including(1) trapping the antigen, and releasing it slowly, (2) stimulatingmigration of cells to the injection site, (3) stimulating or trappinglymphocytes, or stimulating lymphocyte proliferation and (4) improvingantigen dispersion within the patient's body. For example, oils,polymers, mineral salts and liposomes have been used as adjuvants inthis regard. By comparison, immunostimulants are substances that inducea general, temporary increase in a patient's immune response, whetheradministered with the antigen or separately. Typical immunostimulantsare bacterial, such as BCG (an attenuated strain of Mycobacteriumtuberculosis) or a nonviable form of Corynebacterium parvum. By eithermechanism, the adjuvant or immunostimulant serves to enhance the desiredspecific immune response by non-specific means.

A serious drawback of many of the adjuvants currently available is theirtoxicity. In general, the best adjuvants (i.e., those that provide thegreatest enhancement of the desired immune response) are also the mosttoxic. Thus, practitioners must continually balance the level ofstimulation against the toxicity of the adjuvant.

Accordingly, there is a need in the art for the identification ofcompounds that provide a desired enhancement of specific immuneresponses, but with low levels of toxicity. The present inventionfulfills these needs and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compounds and methodsrelating to the Leishmania antigen LbeIF4A or LmeIF4A, which ishomologous to the eukaryotic ribosomal protein eIF4A. In one aspect ofthe invention, methods are provided for enhancing or eliciting an immuneresponse to an antigen and/or an antigen encoded by a DNA vaccine in apatient, comprising administering to a patient an antigen and/or a DNAvaccine, and an LbeIF4A polypeptide comprising an amino acid sequenceencoded by a DNA sequence selected from the group consisting of: (a)nucleotides 115-1323 of SEQ ID NO. 1; and (b) DNA sequences thathybridize to a nucleotide sequence complementary to nucleotides 115-1323of SEQ ID NO. 1 under moderately stringent conditions, wherein the DNAsequence encodes a polypeptide that stimulates a Th1 immune response ina peripheral blood mononuclear cells obtained from a Leishmania-infectedindividual. In another aspect of the invention, methods are provided forenhancing or eliciting an immune response to an antigen and/or anantigen encoded by a DNA vaccine, in a patient, comprising administeringto a patient an antigen and/or a DNA vaccine, and an LmeIF4A polypeptidecomprising an amino acid sequence encoded by a DNA sequence selectedfrom the group consisting of: (a) nucleotides 117 through 1325 of SEQ IDNO:3; and (b) DNA sequences that hybridize to a nucleotide sequencecomplementary to nucleotides 117 through 1325 of SEQ ID NO:3 undermoderately stringent conditions, wherein the DNA sequence encodes apolypeptide that stimulates a Th1 immune response in peripheral bloodmononuclear cells obtained from a Leishmania-infected individual.

In related aspects, the present invention provides methods for enhancingan immune response to an antigen and/or an antigen encoded by a DNAvaccine in a patient, comprising administering to a patient an antigenand/or a DNA vaccine, and an LbeIF4A polypeptide comprising amino acids49-403 of SEQ ID NO: 2, or a variant thereof that differs only inconservative substitutions and/or modifications, or an antigen and/or aDNA vaccine and an LmeIF4A polypeptide comprising amino acids 49-403 ofSEQ ID NO: 4, or a variant thereof that differs only in conservativesubstitutions and/or modifications.

In another related aspect, methods are provided for enhancing an immuneresponse in a biological sample, comprising contacting a biologicalsample with an antigen and/or an antigen encoded by a DNA vaccine, andan LbeIF4A polypeptide as described above, wherein the biological samplecomprises cells selected from the group consisting of peripheral bloodmononuclear cells, monocytes, B cells, dendritic cells, and combinationsthereof.

In yet another related aspect, methods are provided for enhancing oreliciting an immune response in a biological sample, comprisingcontacting a biological sample with an LmeIF4A polypeptide as describedabove, wherein the biological sample comprises cells selected from thegroup consisting of peripheral blood mononuclear cells, monocytes, Bcells, dendritic cells and combinations thereof.

In another aspect, methods are provided for enhancing an immune responseto a tumor in a patient, comprising administering to a patient a tumorantigen or antigens and/or a DNA vaccine, and an LbeIF4A or an LmeIF4Apolypeptide as described above.

Within further aspects, methods are provided for treating a tumor in apatient, comprising administering to a patient an LbeIF4A or LmeIF4Apolypeptide, as described above.

Within each of the aspects noted above, as an alternative to utilizingan LbeIF4A or LmeIF4A polypeptide, one can utilize viral vectors ornucleic acid molecules (collectively, the “nucleic acid compositions”)directing the expression of the polypeptide in patient cells infected ortransfected with the nucleic acid compositions. The step ofadministering the nucleic acid composition may be performed in vivo orex vivo, the latter including the subsequent administration of theinfected/transfected cells. In addition, where an antigen or tumorantigen is administered, it will be evident that the nucleic acidcomposition may also be designed to direct the expression of suchantigens (either on the same or different vectors or molecules).

Within further aspects, methods are provided for treating a Th2-mediateddisease in a patient, comprising administering to a patient (a) anLeIF4A polypeptide; (b) a nucleic acid molecule directing the expressionof an LeIF4A polypeptide in patient cells transfected with the nucleicacid molecule or (c) a viral vector directing the expression of anLeIF4A polypeptide in patient cells infected with the viral vector.Th2-mediated diseases include asthma, allergy, Th2 mediated autoimmunedisease and Helminth infection.

The present invention further provides methods for decreasing productionof one or more Th2-associated cytokines in a patient, comprisingadministering to a patient (a) an LeIF4A polypeptide; (b) a nucleic acidmolecule directing the expression of an LeIF4A polypeptide in patientcells transfected with the nucleic acid molecule or (c) a viral vectordirecting the expression of an LeIF4A polypeptide in patient cellsinfected with the viral vector. Within certain embodiments, theTh2-associated cytokine is IL-4 or IL-5.

Methods for stimulating or enhancing IL-18 production in a patient arealso provided, comprising administering to a patient (a) an LeIF4Apolypeptide; (b) a nucleic acid molecule directing the expression of anLeIF4A polypeptide in patient cells transfected with the nucleic acidmolecule or (c) a viral vector directing the expression of an LeIF4Apolypeptide in patient cells infected with the viral vector.

Within further aspects, methods are provided for enhancing or elicitingan immune response to an antigen in a biological sample, comprisingcontacting a biological sample with an LeIF4A polypeptide in combinationwith one or more Th1-associated cytokines.

The present invention also provides method for enhancing or eliciting animmune response to an antigen in a patient, comprising administering toa patient one or more Th1-associated cytokines in combination with (a)an LeIF4A polypeptide; (b) a nucleic acid molecule directing theexpression of an LeIF4A polypeptide in patient cells transfected withthe nucleic acid molecule or (c) a viral vector directing the expressionof an LeIF4A polypeptide in patient cells infected with the viralvector. The Th1-associated cytokines may be IL-2, IL-12, IL-15 and/orIL-18.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the results of Southern blot analysis of Leishmania spp.DNA, indicating that the Leishmania eIF4A homologue is conserved andthat L. braziliensis genomic DNA contains at least two copies ofLbeIF4A.

FIG. 2 shows the results of an immunoblot analysis which demonstratesthat LbeIF4A immune rabbit serum reacts with one dominant proteinspecies of size ˜45 kDa in different Leishmania species.

FIG. 3 illustrates the ability of purified recombinant LbeIF4A tostimulate proliferation of PBMCs from L. braziliensis-infectedindividuals.

FIGS. 4A and 4B present the results obtained by analysis of cytokinemRNA expression patterns of PBMCs from patients with confirmed cases ofL. braziliensis infection.

FIG. 5 illustrates the supernatant levels of secreted IFN-γ from PBMCsfrom L. braziliensis-infected individuals following stimulation withLbeIF4A or parasite lysate.

FIG. 6 shows the levels of TNF-α detected in the supernatants of PBMCsfrom L. braziliensis-infected individuals following stimulation withLbeIF4A or parasite lysate.

FIG. 7, Panels A-D, shows that LbeIF4A also stimulates patient PBMCs tosecrete IL-12 in the cultured supernatant with a magnitude significantlyhigher than the IL-12 level stimulated by parasite lysate and that IL-10inhibits this IL-12 production.

FIG. 8, Panels A and B, demonstrates that in all patient PBMCs tested,IFN-γ production was IL-12 dependent and inhibited by IL-10.

FIGS. 9A and 9B show that LbeIF4A stimulates IL-12 production incultured human macrophages and adherent PBMCs.

FIG. 10 indicates that LbeIF4A stimulates IL-12 p40 production in thehuman myeloid leukemia cell-line, THP-1, and synergizes with IFN-γ tostimulate THP-1 cells to secrete IL-12.

FIG. 11 presents results that indicate that lymph node cells of miceprimed with LbeIF4A proliferate and secrete an almost exclusive Th1cytokine profile.

FIG. 12 demonstrates that LbeIF4A provides significant protectionagainst L. major infection in an animal model recognized as havingrelevance to human disease.

FIG. 13 illustrates the elicitation of anti-ovalbumin CTL using arepresentative LmeIF4A polypeptide.

FIG. 14 illustrates the use of a representative LmeIF4A polypeptide asan adjuvant for the induction of antibodies specific for trinitrophenol.

FIG. 15 shows the enhancement of anti-MUC-1 antibody production by arepresentative LmeIF4A polypeptide.

FIG. 16 illustrates the enhancement of specific CTL activity by arepresentative LmeIF4A polypeptide in cultured cells.

FIG. 17 shows the in vitro stimulation of CTL activity with IL-2, withand without an LmeIF4A polypeptide.

FIG. 18 illustrates the induction of murine alloreactive CTL by anLmeIF4A polypeptide.

FIG. 19 shows tumor regression following administration of tumor antigenand LmeIF4A polypeptide contained in microspheres.

FIG. 20 shows tumor regression following administration of tumor antigenand LmeIF4A polypeptide contained in microspheres.

FIG. 21 shows tumor regression following administration of solubleLmeIF4A polypeptide and tumor antigen contained in microspheres.

FIG. 22 presents a comparison of the predicted amino acid sequences ofL. major eIF4A (LmeIF), with the homologous proteins from L.brazilienses (LbeIF), mouse (MeIF) and human (HeIF). Positions ofidentical residues to LmeIF are shaded black. Boxed sequences representidentity between the mouse and human proteins that are distinct from theLeishmania homologue or conservative substitutions. Regions ofsimilarity with conserved elements found in RNA helicases are indicated(I-VI). I and II (DEAD) represent specialized versions of the A and Bmotifs described in other ATP binding proteins. Cysteine residues areindicated by * and potential N-linked glycosylation sites areunderlined.

FIGS. 23 A-C illustrate the expression and purification of recombinantLmeIF. FIG. 23A is a photograph of coomassie blue-stained 12% SDS-PAGEof E. coli lysates before (lane 1) and after (lane 2) induction withIPTG to express rLeIF with 6 amino-terminal histidine tag residues.rLmeIF following purification from the inclusion body by affinitychromatigraphy on Ni-NTA column is shown in lane 3. FIG. 23B is aphotograph of coomassie blue-stained 12% SDS-PAGE of overlapping LeIFdeletions. The recombinant clones were designed to encode the N-terminalhalf (26 kDa, residues 1-226, lane 1), the middle portion (16 kDa,residues 129-261, lane 2) and the C-terminal half (25 kDa, residues196-403) of LeIF with six His-tag residues and the proteins purifiedover NiNTA resin. Protein molecular weight markers (lane M) areindicated to the left. FIG. 23C is a schematic representation of thefull length cDNA clone of L. major LeIF comprising of a 0.13 kb sequenceof 5′ untranslated (5′ UTR) segment, an open reading frame of 1.209 kbcoding for 403 amino acid long protein, and a 1.25 kb of 3′ UTRterminating with a stretch of poly A tail. The arrows below show thelocation and sizes of both the full-length and overlapping fragments ofthe LeIF constructs.

FIGS. 24A-C are graphs illustrating the analysis of the Th1/Th2 cytokineprofile of draining lymph node cells from L. major infected BALB/c miceagainst rLeIF. Draining popliteal lymph node cells (2×10⁶/ml) isolatedat (A) 10 and (B), 28 days of infection were stimulated in vitro with 10μg/ml each of rLeIF or SLA and the supernatants analyzed 72 hours laterfor the amount of IFN-γ and IL-4. In FIG. 24C, L. major infection serafrom BALB/c mice (28 day post-infection) were analyzed and titrated forthe presence of anti-rLeIF or rLmSTI1 specific antibody and comparedwith total promastigote lysate (SLA). Bound antibodies were detectedwith HRP conjugated goat anti-mouse IgG secondary antibody.

FIG. 25 is a histogram illustrating the abrogation of the SLA-inducedIL-4 secretion by LeIF. Lymph node cells were obtained from BALB/c miceinfected with L. major (4 weeks post-infection) and were stimulated withSLA (10 μg/ml) alone or in the presence of various concentration ofLeIF. Cells were cultured for 3 days and supernatants were collected andassayed for the production of IL-4 and IFN-γ by ELISA.

FIG. 26 is a histogram illustrating the profile of T cell clonesisolated from rLeIF or r8E-primed BALB/c mice. Mice were immunizedsubcutaneously with 70 μg of the respective antigens without adjuvant.Ten days later, their lymph node cells were restimulated in vitro underlimiting dilution with the same antigen, irradiated antigen presentingcells and IL-2. The resulting clones were re-stimulated with anti-CD3mAb and supernatant cytokine patterns of the Th1- (IFN-γ), Th2- (IL-4)or Th0 (IFN-γ and IL-4) were determined by ELISA. The result ispresented as the percentage of clones expressing a Th1, Th2, or Th0cytokine profile.

FIGS. 27A-C are histograms illustrating LeIF stimulation of theproduction of IFN-γ by splenocytes from naive C3H- and Balb/c-SCID mice.In FIG. 27A, splenocytes from SCID mice of both Balb/c and C3Hbackground were cultured at 2×10⁶ per well and stimulated with 10 μg/mlof the indicated antigen. Supernatants were harvested at 12, 24, and 72hours and assayed for the production of IFN-γ. In FIG. 27B assays wereperformed as above using C3H SCID splenocytes in the presence or absenceof anti-IL-12 antibody. Supernatants were harvested at 72 hours. In FIG.27C, stimulation was performed using three overlapping LeIF recombinantscomprising amino acid residues 1-226, 129-261 and 196-403 at 2.5, 5.0,and 10 μg/ml in splenocyte cultures from C3H SCID mice. As control, LPSwas used at two concentrations, 100 ng and 1 μg/ml.

FIG. 28 is a photograph depicting the electrophoresis of RT-PCRexperiments to determine the presence of various cytokines in SCID mousesplenocytes cultured for 24 hours in the absence (−) or presence (+) ofLeIF(10 μg/ml). Primers were specific for β-actin as a control, or forIFN-γ, IL-18 or IL-10, as indicated.

FIG. 29 is a graph showing the level of IFN-γ in SCID mouse splenocytesstimulated for 72 hours with varying amounts of IL-18 as indicated, inthe presence or absence of LeIF (10 μg/ml).

FIG. 30 is a graph showing the level of IFN-γ in SCID mouse splenocytesstimulated for 72 hours with varying amounts of IL-8 as indicated, inthe presence or absence of LeIF (10 μg/ml), IL-15 (ng/ml) or both.

FIG. 31 is a graph showing the level of IFN-γ in SCID mouse splenocytesstimulated for 72 hours with varying amounts of IL-18 as indicated, inthe presence or absence of LeIF (10 μg/ml), IL-15 (100 ng/ml) or both.

FIG. 32 is a graph illustrating the cytotoxic activity (expressed as %Specific Lysis) of SCID mouse splenocytes stimulated with IL-15 (100ng/ml), IL-12 (10 U/ml) or both in the presence or absence of LeIF (10μg/ml) at varying effector:target ratios, as indicated. The target cellswere YAC-1 cells.

FIG. 33 is a graph illustrating the cytotoxic activity (expressed as %Specific Lysis) of SCID mouse splenocytes stimulated with IL-18 (100ng/ml), IL-12 (10 U/ml) or both in the presence or absence of LeIF (10μg/ml) at varying effector:target ratios, as indicated. The target cellswere YAC-1 cells.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to theenhancement of immune responses, which may be humoral and/orcell-mediated, in a patient or cell culture. Within the context of thisinvention, an immune response to an antigen, including animmunostimulating antigen (i.e., an antigen against which a patientraises an immune response), may be initiated or enhanced byadministering to the patient the antigen and one or more LbeIF4A-derivedor LmeIF4A-derived polypeptides as described herein. Antigens andimmunostimulating antigens are in general protein molecules and includemolecules derived from viruses, such as HIV, HBV, influenza virus,respiratory syncytial virus, bacteria, such as Hemophilus influenza,Pneumoccocus pneumoniae, and parasites, such as Leishmania, andTrypanosoma. In addition, an immune response to a tumor may be enhancedor elicited by administering to the patient a tumor antigen (i.e., anantigen that stimulates an immune response (e.g., CTL) to a tumor).Within the context of this invention, tumor antigens include virallyencoded molecules, MAGE-1, Her-2, PSA, and other molecules. Accordingly,the methods of this invention involve the co-administration of aspecific antigen or immunostimulating antigen and an LeIF4A-derivedpolypeptide as disclosed herein. A tumor may also be treated byadministering to the patient an LbeIF4A or LmeIF4A polypeptide in theabsence of such an exogenously administered tumor antigen.

The LbeIF4A and LmeIF4A polypeptides of the present invention may alsobe used to elicit or enhance an immune response to an antigen encoded bya DNA vaccine. DNA vaccines encode one or more immunostimulatingantigens, such that the antigen is generated in situ. For instance, theDNA vaccine may encode a tumor antigen and, optionally, anLeIF4A-derived polypeptide as described herein. In such vaccines, theDNA may be present within any of a variety of delivery systems known tothose of ordinary skill in the art, including nucleic acid expressionsystems, bacteria and viral expression systems. Appropriate nucleic acidexpression systems contain the necessary DNA sequences for expression inthe patient (such as a suitable promoter). Bacterial delivery systemsinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an epitope of a prostate cellantigen on its cell surface. The DNA may be introduced using a viralexpression system (e.g., vaccinia or other pox virus, retrovirus, oradenovirus), which may involve the use of a non-pathogenic (defective),replication competent virus. Suitable systems are disclosed, forexample, in Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexner et al.,Ann. N.Y Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21,1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973;U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805;Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science252:431-434, 1991; Kolls et al., PNAS 91:215-219, 1994; Kass-Eisler etal., PNAS 90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848,1993; and Guzman et al., Cir. Res. 73:1202-1207, 1993. Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. The DNA may also be “naked,” as described,for example, in published PCT application WO 90/11092, and Ulmer et al.,Science 259:1745-1749, 1993, reviewed by Cohen, Science 259:1691-1692,1993. The uptake of naked DNA may be increased by coating he DNA ontobiodegradable beads, which are efficiently transported into the cells.

The compounds of this invention generally comprise a polypeptide thatstimulates a Th1 or CTL (cytotoxic T lymphocyte) immune response inperipheral blood mononuclear cells (PBMCs). In particular, polypeptidescomprising all or a stimulatory portion of a Leishmania braziliensis orLeishmania major homologue of the eukaryotic ribosomal protein eIF4A aredisclosed. Such proteins may be referred to herein as LbeIF4A andLmeIF4A, or as LbeIF and LmeIF, respectively. As used herein, the term“PBMCs” refers to preparations of nuclear cells that are present inperipheral blood. The term “polypeptide,” in the context of thisinvention, encompasses amino acid chains of any length, including fulllength proteins and portions thereof, wherein amino acid residues arelinked by covalent peptide bonds. Therefore, an “LbeIF4A polypeptide”comprises LbeIF4A, or a portion or other variant thereof that retainsstimulatory activity. Similarly, an “LmeIF4A polypeptide” comprisesLmeIF4A, or a portion or other variant thereof that retains stimulatoryactivity. As used herein, “LeIF4A” or “LeIF” refers to either LbeIF4A orLmeIF4A. Although LbeIF4A is described herein for exemplary purposes,within the context of this invention, LmeIF4A, portions thereof, andvariants of the polypeptide (or portions thereof) may also be used. AnLeIF4A polypeptide may consist entirely of one or more stimulatoryportions of LeIF4A, or the stimulatory portion(s) may be supplied in thecontext of a larger protein that contains additional LeIF4A sequencesand/or amino acid sequences heterologous to LeIF4A. Preferably, thepolypeptides are substantially free of contaminating endogenousmaterials.

The polypeptides of the present invention include variants of LbeIF4A orLmeIF4A that retain the ability to stimulate a Th1 or CTL immuneresponse in PBMCs. Such variants include various structural forms of theprimary protein. Due to the presence of ionizable amino and carboxylgroups, for example, a LbeIF4A polypeptide may be in the form of anacidic or basic salt, or may be in neutral form. Individual amino acidresidues may also be modified by oxidation or reduction.

Variants within the scope of this invention also include polypeptides inwhich the primary amino acid structure of LeIF4A or a fragment thereofis modified by forming covalent or aggregative conjugates with otherpolypeptides or chemical moieties such as glycosyl groups, lipids,phosphate, acetyl groups and the like. Covalent derivatives may beprepared, for example, by linking particular functional groups to aminoacid side chains or at the N- or C-termini. Alternatively, forderivatives in which a polypeptide is joined to a LeIF4A polypeptide, afusion protein may be prepared using recombinant DNA techniques, asdescribed below. In one such embodiment, the LeIF4A polypeptide may beconjugated to a signal (or leader) polypeptide sequence at theN-terminal region of the protein which co-translationally orpost-translationally directs transfer of the protein from its site ofsynthesis to its site of function inside or outside of the cell membraneor wall (e.g., the yeast α-factor leader).

Protein fusions within the present invention may also comprise peptidesadded to facilitate purification or identification of LeIF4Apolypeptides (e.g., poly-His). For example, the peptide described byHopp et al., Bio/Technology 6:1204 (1988) is a highly antigenic peptidethat can be used to facilitate identification. Such a peptide providesan epitope reversibly bound by a specific monoclonal antibody, enablingrapid assay and facile purification of expressed recombinant protein.The sequence of Hopp et al. is also specifically cleaved by bovinemucosal enterokinase, allowing removal of the peptide from the purifiedprotein. Fusion proteins capped with such peptides may also be resistantto intracellular degradation in E. coli.

Protein fusions encompassed by this invention further include, forexample, LeIF4A polypeptides linked to an immunoglobulin Fc region. IfLbeIF4A fusion proteins are made with both heavy and light chains of anantibody, it is possible to form a protein oligomer with as many as fourLbeIF4A protein regions. Also within the scope of the present inventionare LbeIF4A polypeptides linked to a leucine zipper domain. Leucinezipper domains are described, for example, in published PCT ApplicationWO 94/10308. LbeIF4A polypeptides comprising leucine zippers may, forexample, be oligomeric, dimeric or trimeric. All of the above proteinfusions may be prepared by chemical linkage or as fusion proteins, asdescribed below.

Preferred protein fusions include polypeptides that comprise sequencesuseful for stimulating immunity to infectious pathogens (e.g.,antigens). Such sequences may be derived, for example, from viruses,tumor cells, parasites or bacteria.

The present invention also includes LeIF4A polypeptides with or withoutassociated native-pattern glycosylation. Polypeptides expressed in yeastor mammalian expression systems may be similar to or slightly differentin molecular weight and glycosylation pattern than the native molecules,depending upon the expression system. For instance, expression of DNAencoding LbeIF4A polypeptides in bacteria such as E. coli providesnon-glycosylated molecules. N-glycosylation sites of eukaryotic proteinsare characterized by the amino acid triplet Asn-A₁-Z, where A₁ is anyamino acid except Pro, and Z is Ser or Thr. Variants of LbeIF4Apolypeptides having inactivated N-glycosylation sites can be produced bytechniques known to those of ordinary skill in the art, such asoligonucleotide synthesis and ligation or site-specific mutagenesistechniques, and are within the scope of this invention. Alternatively,N-linked glycosylation sites can be added to a LbeIF4A polypeptide.

The polypeptides of this invention also include variants of LeIF4Apolypeptides that have an amino acid sequence different from the nativeLeIF4A protein because of one or more deletions, insertions,substitutions or other modifications. Such variants should besubstantially homologous to the native LeIF4A and should retain theability to stimulate a Th1 or CTL immune response in PBMCs. “Substantialhomology,” as used herein, refers to amino acid sequences that may beencoded by DNA sequences that are capable of hybridizing undermoderately stringent conditions to a naturally occurring DNA sequenceencoding, for instance, LbeIF4A. Suitable moderately stringentconditions include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mMEDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight; followedby washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and0.2×SSC containing 0.1% SDS). Such hybridizing DNA sequences are alsowithin the scope of this invention. The effect of any such modificationson the activity of a LbeIF4A polypeptide may be readily determined byanalyzing the ability of the mutated LbeIF4A peptide to induce a Th1 orCTL response using, for example, any of the methods described herein. Apreferred variant of LbeIF4A is the Leishmania major homologue ofLbeIF4A (LmeIF4A).

Generally, amino acid substitutions should be made conservatively; i.e.,a substitute amino acid should replace an amino acid that has similarproperties, such that one skilled in the art of peptide chemistry wouldexpect the secondary structure and hydropathic nature of the polypeptideto be, substantially unchanged. In general, the following groups ofamino acids represent conservative changes: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Variants within thescope of this invention may also, or alternatively, contain othermodifications, including the deletion or addition of amino acids, thathave minimal influence on the stimulatory properties, secondarystructure and hydropathic nature of the polypeptide. In general,fragments of LeIF4A may be constructed by deleting terminal or internalresidues or sequences. Additional guidance as to suitable modificationsmay be obtained by a comparison of the sequence of LeIF4A to thesequences and structures of other eIF4A family members. For example,terminal or internal residues or sequences of LeIF4A not needed forbiological activity may be deleted. Cysteine residues may be deleted orreplaced with other amino acids to prevent formation of incorrectintramolecular disulfide bridges upon renaturation. Other approaches tomutagenesis involve modification of adjacent dibasic amino acid residuesto enhance expression in yeast systems in which KEX2 protease activityis present.

An LeIF4A full length protein may generally be obtained using a genomicor cDNA clone encoding the protein. A genomic sequence that encodes fulllength LbeIF4A is shown in SEQ ID NO:1, and the deduced amino acidsequence is presented in SEQ ID NO:2. A genomic sequence that encodesfull length LmeIF4A is shown in SEQ ID NO:3 and the deduced amino acidsequence in SEQ ID NO:4. Such clones may be isolated by screening anappropriate Leishmania braziliensis or Leishmania major expressionlibrary for clones that express antigens that react with sera from apatient afflicted with mucosal leishmaniasis, and then analyzing thereactive antigens for the ability to stimulate proliferative responsesand preferential Th1 cytokine production in patient T cell assays or forthe ability to stimulate a CTL response in patient T cells. The librarypreparation and screen may generally be performed using methods known tothose of ordinary skill in the art, such as methods described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratories, Cold Spring Harbor. N.Y., 1989, which isincorporated herein by reference. Briefly, a bacteriophage expressionlibrary may be plated and transferred to filters. The filters may thenbe incubated with serum and a detection reagent. In the context of thisinvention, a “detection reagent” is any compound capable of binding tothe antibody-antigen complex, which may then be detected by any of avariety of means known to those of ordinary skill in the art. Typicaldetection reagents contain a “binding agent,” such as Protein A, ProteinG, IgG or a lectin, coupled to a reporter group. Preferred reportergroups include enzymes, substrates, cofactors, inhibitors, dyes,radionuclides, luminescent groups, fluorescent groups and biotin. Morepreferably, the reporter group is horseradish peroxidase, which may bedetected by incubation with a substrate such as tetramethylbenzidine or2,2′-azino-di-3-ethylbenzthiazoline sulfonic acid. Plaques containinggenomic or cDNA sequences that express a protein which binds to anantibody in the serum are isolated and purified by techniques known tothose of ordinary skill in the art. Appropriate methods may be found,for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989.

Patient T cell assays may generally be performed by treating patientPBMCs with the reactive antigens and analyzing the cells for a suitableresponse. For example, the PBMC supematant may be assayed for the levelof secreted cytokines. Preferably, the cytokine assayed is interferon-γ,interleukin-2, interleukin-12 (either the p40 subunit or biologicallyactive p70), interleukin-1 or tumor necrosis factor-α. The cytokinesinterleukin-4 and interleukin-10 may also be assayed, since the levelsof these representative Th2-type cytokines generally decrease inresponse to treatment with a polypeptide as described herein. Cytokinesmay be assayed, for example, using commercially available antibodiesspecific for the cytokine of interest in an ELISA format, with positiveresults determined according to the manufacturer's instructions.Suitable antibodies may be obtained, for example, from Chemicon,Temucula, Calif. and PharMingen, San Diego, Calif. Alternatively, thetreated PBMCs may be assayed for mRNA encoding one or more of thecytokines interferon-γ, interleukin-2, interleukin-12 p40 subunit,interleukin-1 or tumor necrosis factor-α, or the PBMCs may be assayedfor a proliferative response as described herein. Alternatively,cytokines may be measured by testing PBMC supernatants forcytokine-specific biological activities.

Variants of LeIF4A that retain the ability to stimulate a Th1 immuneresponse in PBMCs may generally be identified by modifying the sequencein one or more of the aspects described above and assaying the resultingpolypeptide for the ability to stimulate a Th1 response. Such assays maygenerally be performed by treating patient PBMCs with the modifiedpolypeptide and assaying the response, as described above. Naturallyoccurring variants of LeIF4A may also be isolated from other Leishmaniaspecies by, for example, screening an appropriate cDNA or genomiclibrary with a DNA sequence encoding LeIF4A or a variant thereof.

The above-described sequence modifications may be introduced usingstandard recombinant techniques or by automated synthesis of themodified polypeptide. For example, mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analogue having the desired amino acid insertion,substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide a gene in which particular codonsare altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al., Gene 42:133, 1986; Bauer et al., Gene37:73, 1985; Craik, BioTechniques, Jan. 12-19, 1985; Smith et al.,Genetic Engineering: Principles and Methods, Plenum Press, 1981; andU.S. Pat. Nos. 4,518,584 and 4,737,462.

Mutations in nucleotide sequences constructed for expression of suchLeIF4A polypeptides must, of course, preserve the reading frame of thecoding sequences and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structures, such as loopsor hairpins, which would adversely affect translation of the receptormRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed LeIF4A protein mutants screened for the desired activity.

Not all mutations in a nucleotide sequence which encodes a LeIF4Aprotein will be expressed in the final product. For example, nucleotidesubstitutions may be made to enhance expression, primarily to avoidsecondary structure loops in the transcribed mRNA (see, e.g., EuropeanPatent Application 75,444A), or to provide codons that are more readilytranslated by the selected host, such as the well-known E. colipreference codons for E. coli expression.

The polypeptides of the present invention, both naturally occurring andmodified, are preferably produced by recombinant DNA methods. Suchmethods include inserting a DNA sequence encoding a LeIF4A polypeptideinto a recombinant expression vector and expressing the DNA sequence ina recombinant microbial, mammalian or insect cell expression systemunder conditions promoting expression. DNA sequences encoding thepolypeptides provided by this invention can be assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene which is capable of beinginserted in a recombinant expression vector and expressed in arecombinant transcriptional unit.

Recombinant expression vectors contain a DNA sequence encoding a LeIF4Apolypeptide operably linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. Such regulatory elements include a transcriptional promoter, anoptional operator sequence to control transcription, a sequence encodingsuitable mRNA ribosomal binding sites, and sequences which control thetermination of transcription and translation, as described in detailbelow. An origin of replication and a selectable marker to facilitaterecognition of transformants may additionally be incorporated.

DNA regions are operably linked when they are functionally related toeach other. For example, DNA for a signal peptide (secretory leader) isoperably linked to DNA for a polypeptide if it is expressed as aprecursor which participates in the secretion of the polypeptide; apromoter is operably linked to a coding sequence if it controls thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation. Generally, operably linked means contiguous and, in thecase of secretory leaders, in reading frame. DNA sequences encodingLeIF4A polypeptides which are to be expressed in a microorganism willpreferably contain no introns that could prematurely terminatetranscription of DNA into mRNA.

Expression vectors for bacterial use may comprise a selectable markerand bacterial origin of replication derived from commercially availableplasmids comprising genetic elements of the well known cloning vectorpBR322 (ATCC 37017). Such commercial vectors include, for example,pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and PGEM1 (PromegaBiotec, Madison, Wis., USA). These pBR322 “backbone” sections arecombined with an appropriate promoter and the structural sequence to beexpressed. E. coli is typically transformed using derivatives of pBR322,a plasmid derived from an E. coli species (Bolivar et al., Gene 2:95,1977). pBR322 contains genes for ampicillin and tetracycline resistanceand thus provides simple means for identifying transformed cells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and European Patent Application 36,776) and the tacpromoter (Maniatis, Molecular Cloning. A Laboratory Manual, Cold SpringHarbor Laboratory, p.412, 1982). A particularly useful bacterialexpression system employs the phage λ P_(L) promoter and cI857tsthermolabile repressor. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2, resident in E. coli strain JMB9 (ATCC 37092) andpPLc28, resident in E. coli RR1 (ATCC 53082).

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., European Patent Application 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed (see, e.g., Kurjan et al., Cell30:933, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,1984). The leader sequence may be modified to contain, near its 3′ end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes. The transcriptional and translational controlsequences in expression vectors to be used in transforming vertebratecells may be provided by viral sources. For example, commonly usedpromoters and enhancers are derived from polyoma, adenovirus 2, simianvirus 40 (SV40), and human cytomegalovirus. DNA sequences derived fromthe SV40 viral genome, for example, SV40 origin, early and latepromoter, enhancer, splice, and polyadenylation sites may be used toprovide the other genetic elements required for expression of aheterologous DNA sequence. The early and late promoters are particularlyuseful because both are obtained easily from the virus as a fragmentwhich also contains the SV40 viral origin of replication (Fiers et al.,Nature 273:113, 1978). Smaller or larger SV40 fragments may also beused, provided the approximately 250 bp sequence extending from theHindIII site toward the BglII site located in the viral origin ofreplication is included. Further, viral genomic promoter, control and/orsignal sequences may be utilized, provided such control sequences arecompatible with the host cell chosen. Exemplary vectors can beconstructed as disclosed by Okayama and Berg, Mol. Cell. Biol. 3:280,1983.

A useful system for stable high level expression of mammalian receptorcDNAs in C127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A preferred eukaryotic vector for expression of LbeIF4A proteinDNA is pDC406 (McMahan et al., EMBO J. 10:2821, 1991), and includesregulatory sequences derived from SV40, human immunodeficiency virus(HIV), and Epstein-Barr virus (EBV). Other preferred vectors includepDC409 and pDC410, which are derived from pDC406. pDC410 was derivedfrom pDC406 by substituting the EBV origin of replication with sequencesencoding the SV40 large T antigen. pDC409 differs from pDC406 in that aBglII restriction site outside of the multiple cloning site has beendeleted, making the BglII site within the multiple cloning site unique.

A useful cell line that allows for episomal replication of expressionvectors, such as pDC406 and pDC409, which contain the EBV origin ofreplication, is CV-1/EBNA (ATCC CRL 10478). The CV-L/EBNA cell line wasderived by transfection of the CV-1 cell line with a gene encodingEpstein-Barr virus nuclear antigen-I (EBNA-1) and constitutively expressEBNA-1 driven from human CMV immediate-early enhancer/promoter.

Transformed host cells are cells which have been transformed ortransfected with expression vectors constructed using recombinant DNAtechniques and which contain sequences encoding a LeIF4A polypeptide ofthe present invention. Transformed host cells may express the desiredLeIF4A polypeptide, but host cells transformed for purposes of cloningor amplifying LeIF4A DNA do not need to express the LeIF4A protein.Expressed LeIF4A proteins will preferably be secreted into the culturesupernatant, depending on the DNA selected, but may also be deposited inthe cell membrane.

Suitable host cells for expression of recombinant proteins includeprokaryotes, yeast or higher eukaryotic cells under the control ofappropriate promoters. Prokaryotes include gram negative or grampositive organisms, for example E. coli or Bacilli. Higher eukaryoticcells include established cell lines of insect or mammalian origin asdescribed below. Cell-free translation systems could also be employed toproduce LbeIF4A proteins using RNAs derived from the DNA constructsdisclosed herein. Appropriate cloning and expression vectors for usewith bacterial, fungal, yeast, and mammalian cellular hosts aredescribed, for example, by Pouwels et al., Cloning Vectors: A LaboratoryManual, Elsevier, N.Y., 1985.

Prokaryotic expression hosts may be used for expression of LeIF4Apolypeptides that do not require extensive proteolytic and disulfideprocessing. Prokaryotic expression vectors generally comprise one ormore phenotypic selectable markers, for example a gene encoding proteinsconferring antibiotic resistance or supplying an autotrophicrequirement, and an origin of replication recognized by the host toensure amplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium, and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although other hosts may also beemployed.

Recombinant LeIF4A polypeptides may also be expressed in yeast hosts,preferably from the Saccharomyces species, such as S. cerevisiae. Yeastof other genera, such as Pichia or Kluyveromyces may also be employed.Yeast vectors will generally contain an origin of replication from the2μ yeast plasmid or an autonomously replicating sequence (ARS), apromoter, DNA encoding the LeIF4A polypeptide, sequences forpolyadenylation and transcription termination and a selection gene.Preferably, yeast vectors will include an origin of replication andselectable marker permitting transformation of both yeast and E. coli,e.g., the ampicillin resistance gene of E. coli and the S. cerevisiaetrp1 gene, which provides a selection marker for a mutant strain ofyeast lacking the ability to grow in tryptophan, and a promoter derivedfrom a highly expressed yeast gene to induce transcription of astructural sequence downstream. The presence of the trp1 lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

Suitable yeast transformation protocols are known to those of skill inthe art. An exemplary technique described by Hind et al. (Proc. Natl.Acad. Sci. USA 75:1929, 1978), involves selecting for Trp⁺ transformantsin a selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 mg/ml adenine and 20 mg/ml uracil. Hoststrains transformed by vectors comprising the ADH2 promoter may be grownfor expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/mluracil. Derepression of the ADH2 promoter occurs upon exhaustion ofmedium glucose. Crude yeast supernatants are harvested by filtration andheld at 4° C. prior to further purification.

Various mammalian or insect (e.g., Spodoptera or Trichoplusia) cellculture systems can also be employed to express recombinant protein.Baculovirus systems for production of heterologous proteins in insectcells are reviewed, for example, by Luckow and Summers, Bio/Technology6:47, 1988. Examples of suitable mammalian host cell lines include theCOS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175,1981), and other cell lines capable of expressing an appropriate vectorincluding, for example, CV-1/EBNA (ATCC CRL 10478), L cells, C127, 3T3,Chinese hamster ovary (CHO), COS, NS-1, HeLa and BHK cell lines.Mammalian expression vectors may comprise nontranscribed elements suchas an origin of replication, a suitable promoter and enhancer linked tothe gene to be expressed, and other 5′ or 3′ flanking nontranscribedsequences, and 5′ or 3′ nontranslated sequences, such as necessaryribosome binding sites, a polyadenylation site, splice donor andacceptor sites, and transcriptional termination sequences.

Purified LeIF4A polypeptides may be prepared by culturing suitablehost/vector systems to express the recombinant translation products ofthe DNAs of the present invention, which are then purified from culturemedia or cell extracts. For example, supernatants from systems whichsecrete recombinant protein into culture media may be first concentratedusing a commercially available protein concentration filter, such as anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate may be applied to a suitablepurification matrix. For example, a suitable affinity matrix maycomprise a counter structure protein (i.e., a protein to which LeIF4Abinds in a specific interaction based on structure) or lectin orantibody molecule bound to a suitable support. Alternatively, an anionexchange resin can be employed, for example, a matrix or substratehaving pendant diethylaminoethyl (DEAE) groups. The matrices can beacrylamide, agarose, dextran, cellulose or other types commonly employedin protein purification. Alternatively, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups arepreferred. Gel filtration chromatography also provides a means ofpurifying LeIF4A.

Affinity chromatography is a particularly preferred method of purifyingLeIF4A polypeptides. For example, a LeIF4A polypeptide expressed as afusion protein comprising an immunoglobulin Fc region can be purifiedusing Protein A or Protein G affinity chromatography. Moreover, a LeIF4Aprotein comprising a leucine zipper domain may be purified on a resincomprising an antibody specific to the leucine zipper domain. Monoclonalantibodies against the LeIF4A protein may also be useful in affinitychromatography purification, by utilizing methods that are well-known inthe art.

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify a LbeIF4A protein composition. Some or allof the foregoing purification steps, in various combinations, can alsobe employed to provide a homogeneous recombinant protein.

Recombinant LeIF4A polypeptide produced in bacterial culture ispreferably isolated by initial extraction from cell pellets, followed byone or more concentration, salting-out, aqueous ion exchange or sizeexclusion chromatography steps. High performance liquid chromatography(HPLC) may be employed for final purification steps. Microbial cellsemployed in expression of recombinant LeIF4A protein can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express LeIF4A polypeptide as a secretedprotein greatly simplifies purification. Secreted recombinant proteinresulting from a large-scale fermentation can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,1984). This reference describes two sequential, reverse-phase HPLC stepsfor purification of recombinant human GM-CSF on a preparative HPLCcolumn.

Preparations of LeIF4A polypeptides synthesized in recombinant culturemay contain non-LeIF4A cell components, including proteins, in amountsand of a character which depend upon the purification steps taken torecover the LeIF4A protein from the culture. These components ordinarilywill be of yeast, prokaryotic or non-human eukaryotic origin. Suchpreparations are typically free of other proteins which may be normallyassociated with the LeIF4A protein as it is found in nature in itsspecies of origin.

Automated synthesis provides an alternate method for preparingpolypeptides of this invention having fewer than about 100 amino acids,and typically fewer than about 50 amino acids. For example, any of thecommercially available solid-phase techniques may be employed, such asthe Merrifield solid phase synthesis method, in which amino acids aresequentially added to a growing amino acid chain. (See Merrifield, J.Am. Chem. Soc. 85:2149-2146, 1963.) Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as AppliedBiosystems, Inc. of Foster City, Calif., and may generally be operatedaccording to the manufacturer's instructions.

As an alternative to the presentation of LeIF4A polypeptides, thesubject invention includes compositions capable of delivering nucleicacid molecules encoding an LeIF4A polypeptide or portion thereof. Suchcompositions include recombinant viral vectors (e.g., retroviruses (seeWO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698, and WO 94/03622),adenovirus (see Berkner, Biotechniques 6:616-627, 1988; Li et al., Hum.Gene Ther. 4:403-409, 1993; Vincent et al., Nat. Genet. 5:130-134, 1993;and Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994), poxvirus (see U.S. Pat. No. 4,769,330; U.S. Pat. No. 5,017,487; and WO89/01973)), naked DNA (see WO 90/11092), nucleic acid molecule complexedto a polycationic molecule (see WO 93/03709), and nucleic acidassociated with liposomes (see Wang et al., Proc. Natl. Acad. Sci. USA84:7851, 1987). In certain embodiments, the DNA may be linked to killedor inactivated adenovirus (see Curiel et al., Hum. Gene Ther. 3:147-154,1992; Cotton et al., Proc. Natl. Acad Sci. USA 89:6094, 1992). Othersuitable compositions include DNA-ligand (see Wu et al., J. Biol. Chem.264:16985-16987, 1989) and lipid-DNA combinations (see Feigner et al.,Proc. Natl. Acad Sci. USA 84:7413-7417, 1989). In addition, theefficiency of naked DNA uptake into cells may be increased by coatingthe DNA onto biodegradable latex beads.

In addition to direct in vivo procedures, ex vivo procedures may be usedin which cells are removed from an animal, modified, and placed into thesame or another animal. It will be evident that one can utilize any ofthe compositions noted above for introduction of LeIF4A nucleic acidmolecules into tissue cells in an ex vivo context. Protocols for viral,physical and chemical methods of uptake are well known in the art.

As noted above, the subject invention provides methods of using thepolypeptides or related nucleic acid compositions disclosed herein forenhancing or eliciting immune responses. It has been found within thepresent invention that LeIF4A contains epitope(s) that stimulateproliferation of PBMCs from Leishmania-infected individuals. LbeIF4Aalso stimulates PBMCs from infected individuals to generate an exclusiveTh1 cytokine profile. A Th1 response is characterized by the productionof the cytokines interleukin-1 (IL-1), interleukin-2 (IL-2),interleukin-12 (IL-12) or interferon-γ (IFN-γ), as well as tumornecrosis factor-α (TNF-α). IL-12 is a heterodimeric molecule comprisingp40 and p35 subunits, which must be coexpressed for the production ofbiologically active IL-12 p70. The p40 subunit is produced only byIL-12-producing cells and is induced in vitro and in vivo afterbacterial and parasite stimulation, whereas the p35 subunit is bothubiquitous and constitutively expressed. Therefore, cells producingIL-12 also have a large excess (10-100 fold) of biologically inactivefree p40 chains. The stimulation of IL-12 production is particularlysignificant as this cytokine has the ability to influence T cellstowards a Th1 response (IFN-γ and IL-2 production). The ability of aprotein to stimulate IL-12 production is therefore an important adjuvantproperty.

LeIF4A also stimulates a Th1 profile of mRNAs encoding IFN-γ, IL-2,IL-12 p40 subunit, and TNF-γ, in PBMCs from Leishmania infectedpatients. No detectable IL-4 or IL-10 mRNA, indicative of a Th2response, is present in such stimulated PBMCs. In fact, LeIF4A generallydown-regulates the expression of such Th2-associated cytokines. Inaddition, LeIF4A stimulates expression of IL-18 mRNA. These propertiesof LeIF4A suggest a role for LeIF4A in generating a protective ortherapeutic immune response in leishmaniasis patients.

In addition, LeIF4A stimulates the production of IL-12 and IL-2 in PBMCsobtained from uninfected control individuals, as well as in culturedhuman macrophages, in the human myeloid leukemia cell line THP-1 and inmice. LeIF4A also synergizes with IFN-γ to stimulate THP-1 cells tosecrete IL-12, and the induction of IFN-γ production by patient PBMCs isabrogated by the presence of anti-IL-12 antibody. The ability tostimulate IL-12 and IL-2 production indicates that LeIF4A has theability to induce an immune response, and that the polypeptidesdescribed herein have a wide applicability in the non-specificenhancement of immune responses.

Accordingly, the present invention discloses methods for enhancing oreliciting, in a patient or cell culture, a cellular immune response(e.g., the generation of antigen-specific cytolytic T cells). Thepresent invention also discloses methods for enhancing or eliciting ahumoral immune response to an antigen (e.g., antigen-reactive antibodyproduction) using a LeIF4A polypeptide (i.e., LbeIF4A, LmeIF4A or avariant thereof) as described above. As used herein, the term “patient”refers to any warm-blooded animal, preferably a human. A patient may beafflicted with a disease, such as leishmaniasis (or other infectiousdiseases) or cancer, such as melanoma, breast cancer, prostate cancer,lymphoma, colon cancer or other tumor, or may be normal (i.e., free ofdetectable disease and infection). A patient may also, or alternatively,be afflicted with any Th2-mediated disease including, but not limitedto, asthma, allergy, Th2-mediated autoimmune disease or Helminthinfection. A “cell culture” is any preparation of PBMCs or isolatedcomponent cells (including, but not limited to, macrophages, monocytes,B cells and dendritic cells). Such cells may be isolated by any of avariety of techniques well known to those of ordinary skill in the art(such as Ficoll-hypaque density centrifugation). The cells may (but neednot) have been isolated from a patient afflicted with leishmaniasis, oranother disorder, and may be reintroduced into a patient aftertreatment.

Within these methods, the LeIF4A polypeptide (or nucleic acidcomposition) is administered to a patient or cell culture along with anantigen, such that it functions as an immunomodulating agent to enhanceor elicit the immune response to the antigen. Within certainembodiments, one or more Th1-associated cytokines (e.g., IL-2. IL-12,IL-15 and/or IL-18) may also be administered in combination with theLeIF4A polypeptide. The LeIF4A polypeptide may be administered withinthe same preparation (e.g., vaccine) as the antigen, or may beadministered separately. In one embodiment, the antigen and the LeIF4Apolypeptide are administered to a patient at the same time and site. Inthis manner, LeIF4A polypeptides may be used, for example, as adjuvantsin vaccine preparations for heterologous agents. In another embodiment,the antigen and LeIF4A polypeptide are administered at different siteson the patient. For example, the LeIF4A polypeptide could beadministered (e.g., injected) in one arm, and the antigen administeredin the other arm. Such administrations may, but need not, take place atthe same time. Alternatively, the LeIF4A polypeptide may be administeredbefore or after the antigen. For example, the LeIF4A polypeptide couldbe administered 24 hours prior to antigen administration. Suitable dosesand methods of administration are presented in detail below.

The immune response generated by a patient to whom a LeIF4A polypeptideis administered may vary, depending on the condition of the patient. ForLeishmania-infected patients, the immune responses that may be generatedinclude a preferential Th1 immune response (which includes stimulationof IL-12 production) and the down-regulation of expression ofTh2-associated cytokines, such as IL-4, IL-5 and/or IL-10. Foruninfected individuals, the immune response may be the production ofIL-12, the production of IL-2, the stimulation of gamma T cells, theproduction of interferon, the generation of antigen-reactive CTL, theproduction of antigen-specific antibodies or any combination thereof.Either type of response provides enhancement of the patient's immuneresponse to the antigen administered with the LeIF4A polypeptide. Inaddition, for patients with diagnosed cancer, such as melanoma, breastcancer, lymphoma, colon cancer, prostate cancer and the like, the immuneresponse may include a preferential CTL response. For treatment of atumor, the immune response should result in a reduction in tumor mass.

The LeIF4A polypeptide (or nucleic acid composition) is preferablyformulated for use in the above methods as a pharmaceutical compositionor a vaccine. Pharmaceutical compositions generally comprise one or moreLbeIF4A polypeptides in combination with a physiologically acceptablecarrier, excipient or diluent. Such carriers will be nontoxic torecipients at the dosages and concentrations employed. The vaccinescomprise one or more LbeIF4A polypeptides and one or more additionalantigens appropriate for the indication. The use of LbeIF4A proteins inconjunction with soluble cytokine receptors, cytokines, andchemotherapeutic agents is also contemplated.

Routes and frequency of administration and polypeptide (or nucleic acidcomposition) doses will vary from individual to individual and mayparallel those currently being used in immunization or treatment ofother infections. In general, the pharmaceutical compositions andvaccines may be administered by injection (e.g., intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. The amount and frequency of administration will depend, ofcourse, on such factors as the nature and severity of the indicationbeing treated, the desired response, the condition of the patient, andso forth. Typically, between 1 and 4 doses may be administered for a 2-6week period. Preferably, two doses are administered, with the seconddose 2-4 weeks later than the first. A suitable dose is an amount ofLeIF4A polypeptide that stimulates the production of IL-12 in thepatient, such that the amount of IL-12 in supernatants of PBMCs isolatedfrom the patient is between about 10 ng and 10 μg per mL. In general,the amount of IL-12 may be determined using any appropriate assay knownto those of ordinary skill in the art, including the assays describedherein. The amount of LbeIF4A polypeptide present in a dose typicallyranges from about 1 pg to about 100 mg per kg of host, typically fromabout 10 pg to about 1 mg, and preferably from about 100 pg to about 1μg. Suitable dose sizes will vary with the size of the animal, but willtypically range from about 0.01 mL to about 5 mL for 10-60 kg animal.Specific appropriate dosages for a particular indication can be readilydetermined.

Alternatively, cells, preferably peripheral blood mononuclear cells, areremoved from a patient and stimulated in vitro with one of the LeIF4Apolypeptides and an antigen (including a tumor antigen). Upon generationof an antigen-specific immune response, such as a CTL response, thecells may be expanded and reinfused into the patient.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administrationand whether a sustained release administration is desired. Forparenteral administration, such as subcutaneous injection, the carrierpreferably comprises water, saline, alcohol, a fat, a wax or a buffer.For oral administration, any of the above carriers or a solid carrier,such as mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, glucose, sucrose, and magnesiumcarbonate, may be employed. Biodegradable microspheres (e.g., polylacticgalactide) may also be employed as carriers for the pharmaceuticalcompositions of this invention. Suitable biodegradable microspheres aredisclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109 and inU.S. patent application Ser. Nos. 08/116,484 and 08/116,802(incorporated by reference herein). The polypeptide orpolypeptide/antigen combination may be encapsulated within thebiodegradable microsphere or associated with the surface of themicrosphere. In this regard, it is preferable that the microsphere belarger than approximately 25 microns.

Pharmaceutical compositions and vaccines may also contain diluents suchas buffers, antioxidants such as ascorbic acid, low molecular weight(less than about 10 residues) polypeptides, proteins, amino acids,carbohydrates including glucose, sucrose or dextrins, chelating agentssuch as EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with nonspecific serum albumin areexemplary appropriate diluents. Preferably, product is formulated as alyophilizate using appropriate excipient solutions (e.g. sucrose) asdiluents.

Optionally, any of a variety of additional agents may be employed in thevaccines or pharmaceutical compositions of this invention, in additionto the LeIF4A polypeptide, to further nonspecifically enhance the immuneresponse. Such agents usually contain a substance designed to protectthe antigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a nonspecific stimulator of immune responses, such as lipid A,Bortadella pertussis or Mycobacterium tuberculosis. Such agents arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.) and MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.).

The following examples are offered by way of illustration, and not byway of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein.

EXAMPLES Example 1 Preparation of DNA Encoding LbeIF4A

This example illustrates the molecular cloning of a DNA sequenceencoding the L. braziliensis ribosomal antigen LbeIF4A.

A genomic expression library was constructed with sheared DNA from L.braziliensis (MHOM/BR/75/M2903) in bacteriophage λZAPII (Stratagene, LaJolla, Calif.). The expression library was screened with E.coli-preadsorbed patient sera from an L. braziliensis-infectedindividual with mucosal leishmaniasis. Plaques containing immunoreactiverecombinant antigens were purified, and the pBSK(−) phagemid excisedusing the manufacturer's protocols. Nested deletions were performed withExonuclease III to generate overlapping deletions for single strandedtemplate preparations and sequencing. Single stranded templates wereisolated following infection with VCSM 13 helper phage as recommended bythe manufacturer (Stratagene, La Jolla, Calif.) and sequenced by thedideoxy chain terminator method or by the Taq dye terminator systemusing the Applied Biosystems Automated Sequencer Model 373A.

The immunoreactive recombinant antigens were then analyzed in patient Tcell assays for their ability to stimulate a proliferative response, asdescribed in Example 5, below, and a dominant Th1 cytokine profile, asdescribed in Example 7, below.

A recombinant clone was identified in the above assays which, followingsequence comparison of its predicted amino acid sequence with sequencesof other proteins, was identified as a Leishmania braziliensis homologueof the eukaryotic initiation factor 4A (eIF4A). The isolated clone(pLeIF.1) lacked the first 48 amino acid residues (144 nucleotides) ofthe full length protein sequence. The pLeIF.1 insert was subsequentlyused to isolate the full length genomic sequence.

SEQ ID NO:1 shows the entire nucleotide sequence of the full-lengthLbeIF4A polypeptide. The open reading frame (nucleotides 115 to 1323)encodes a 403 amino acid protein with a predicted molecular weight of45.3 kD. A comparison of the predicted protein sequence of LbeIF4A withthe homologous proteins from tobacco (TeIF4A), mouse (MeIF4A), and yeast(YeIF4A) shows extensive sequence homology, with the first 20-30 aminoacids being the most variable. The lengths (403, 413, 407, and 395 aminoacids), molecular weights (45.3, 46.8, 46.4, and 44.7 kDa), andisoelectric points (5.9, 5.4, 5.5, and 4.9) of LbeIF4A, TeIF4A, MeIF4Aand YeIF4A, respectively, are similar. LbeIF4A shows an overall homologyof 75.5% (57% identity, 18.5% conservative substitution) with TeIF4A,68.6% (50% identity, 18.6% conservative substitution) with MeIF4A and67.2% (47.6% identity, 19.6% conservative substitution) with YeIF4A.

Example 2 Characterization of the LbeIF4A Gene

This example describes a Southern blot analysis of LbeIF4A DNA inLeishmania species. Leishmania braziliensis (MHOM/BR/75/M2903), L.guyanensis (MHOM/BR/75/M4147) L. amazonensis (IFLA/BR/67/PH8), L.chagasi (MHOM/BR/82/BA-2,Cl and MHOM/BR/84/Jonas), L. donovani(MHOM/Et/67/HU3), L. infantum (IPT-1), L. major (LTM p-2), L. tropica(1063C), Trypanosoma cruzi (MHOM/CH/00/Tulahuen C2) and T. brucei (TREU667) were used and have been previously described (see, Burns et al.,Proc. Natl. Acad Sci. U.S.A. 90:775-779, 1993). Promastigotes andepimastigotes were cultured in axenic media. L. chagasi and L.amazonensis amastigotes were obtained from spleens of Syrian hamstersand footpads of BALB/c ByJ mice respectively, and purified as describedin Burns et al., J. Immunol. 146:742-748, 1991.

Genomic DNA was prepared, digested with enzymes which cut both within(PstI and NotI) and outside of LbeIF4A (BamHI, EcoRI, EcoRV, HindIII,PvuII, and SstI), separated on 0.7% agarose gel and blotted onto Nytran(nylon) membrane, as described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989.A restriction fragment comprising a ˜0.94 kb fragment (nucleotides 143to 1083) of the coding region of LbeIF4A was radiolabeled by the randompriming method (see, Feinberg and Vogelstein, Anal. Biochem.137:266-268, 1984) and blots were hybridized overnight at 65° C. Blotswere washed twice at 65° C. for 20 minutes with each of 2×, 0.5× and0.2×SSC containing 0.1% SDS. L. braziliensis genomic DNA contained atleast two copies of LbeIF4A as exemplified by the presence of twohybridizing bands in the BamHI and PvuII lanes (FIG. 1).

The same figure also illustrates the cross-species conservation betweenthe eIF4A homologue of L. braziliensis and other Leishmania species. Twomajor PstI hybridizing fragments were detected in all other Leishmaniaspecies tested with members of the L. donovani complex (L. chagasi, L.donovani, and L. infantum) showing identical hybridization patterns.LbeIF4A also cross-hybridizes with the more distantly related parasiteT. cruzi but not T. brucei under stringent hybridization conditions.These data show extensive cross-species conservation of the LeishmaniaeIF4A homologue.

Example 3 Preparation of LbeIF4A

This example illustrates the expression and purification of the ˜45 kDaLbeIF4A antigen gene product. The 45 kDa recombinant antigen of thegenomic clone pLeIF.1 (i.e., the antigen lacking the N-terminal 48residues) was purified from 500 ml of IPTG-induced cultures. Theinclusion bodies were isolated and sequentially washed in 10 ml TNE (50mM Tris, pH 8.0. 100 mM NaCl and 10 mM EDTA) containing 2, 4 and 8 Murea. Fractions containing solubilized recombinant antigen (usually the4 and 8 M urea supernatants) were pooled, dialyzed against Tris-bufferedsaline (TBS) and concentrated by precipitation with 30% ammoniumsulfate. Purification to homogeneity was accomplished by preparativeSDS-PAGE electrophoresis, followed by excision and electroelution of therecombinant antigens. All antigens used in our studies had less than 10pg/ml or 1 ng/mg protein endotoxin in a Limulus amebocyte assayperformed by Immunex Corp., Seattle, Wash. These amounts of endotoxinare insignificant for cytokine induction and/or adjuvant activity.

The recombinant antigen was used to immunize a rabbit for the productionof a polyclonal anti-serum. An adult rabbit (New Zealand White; R & RRabbitry, Stanwood, Wash.) was immunized by subcutaneous immunizationwith 100 μg of purified LbeIF4A in incomplete Freund's adjuvant (IFA,GIBCO, Grand Island, N.Y.) together with 100 μg of muramyl dipeptide(adjuvant peptide, Calbiochem-Novabiochem Corp., La Jolla, Calif.),followed by a boost four weeks later with 100 μg of the recombinantantigen in IFA alone. Three weeks later, the rabbit was boostedintravenously with 25 μg of LbeIF4A in saline and serum was collectedone week later.

Immunoblots of L. braziliensis lysates from promastigotes harvestedduring the early-, mid-, or late-log phases or following a temperatureshift of the culture from 22-35° C. were subsequently performed with thepolyclonal rabbit anti-serum as a probe (FIG. 2). Panel A of FIG. 2shows the immunoblot analysis of molecular weight markers (lane M), E.coli lysates from uninduced (lane 1) and induced (lane 2) cultures, andthe purified recombinant antigen (lane 3). Panel B of FIG. 2 shows theimmunoblot analysis of L. braziliensis promastigote lysate (lane 1), L.chagasi promastigote lysate (lane 2), and L. amazonensis promastigote(lane 3) or amastigote (lane 4) lysate.

Parasite and mammalian cell lysates were prepared by freeze/thaw lysisof pellets in SDS sample buffer without glycerol and P-mercaptoethanol.Insoluble material was separated from the supernatant by centrifugationat 10K rpm in a microfuge. Protein concentrations were determined usingthe Pierce BCA protein assay kit. Five to 10 μg of parasite or cellextracts or 0.5 to 1.0 μg of recombinant antigens were separated on12.5% SDS-PAGE and transferred electrophoretically to nitrocellulosemembranes. Reactivities of the antisera were assessed as previouslydescribed (Skeiky et al., J. Exp. Med. 176:201-211, 1992) using[¹²⁵I]-Protein A, followed by autoradiography.

The rabbit anti-serum detected one dominant protein species of size ˜45kD. The relative intensities of the 45 kD eIF4A homologue were similarfor all the lysates analyzed, thus suggesting that this antigen isconstitutively expressed during the early- to mid-log growth phase ofthe parasite or following a temperature transition that mimics theintracellular amastigote stage. This is unlike members of the Leishmaniaheat-shock protein family whose products are upregulated following atemperature transition from 22-35° C. The pre-immune rabbit serum didnot react with the parasite lysates.

Example 4 Preparation of Monoclonal Antibodies That Bind to LbeIF4A

This example illustrates the preparation of monoclonal antibodiesagainst LbeIF4A. Preparations of purified recombinant LbeIF4A ortransfected cells expressing high levels of LbeIF4A, may be employed togenerate monoclonal antibodies against LbeIF4A using conventionaltechniques, such as those disclosed in U.S. Pat. No. 4,411,993. Suchantibodies may be used to interfere with LbeIF4A activation of PBMCs, ascomponents of diagnostic or research assays for LbeIF4A, or in affinitypurification of LbeIF4A.

To immunize rodents, LbeIF4A immunogen is emulsified in an adjuvant(such as complete or incomplete Freund's adjuvant, alum, or anotheradjuvant, such as Ribi adjuvant R700 (Ribi, Hamilton, Mont.), andinjected in amounts ranging from 10-100 μg subcutaneously into aselected rodent, for example, BALB/c mice or Lewis rats. Ten days tothree weeks days later, the immunized animals are boosted withadditional immunogen and periodically boosted thereafter on a weekly,biweekly or every third week immunization schedule. Serum samples areperiodically taken by retro-orbital bleeding or tail-tip excision fortesting by dot-blot assay (antibody sandwich) or ELISA (enzyme-linkedimmunosorbent assay). Other assay procedures are also suitable, such asinhibition of the elicitation of a Th1 response.

Following detection of an appropriate antibody titer, positive animalsare given an intravenous injection of antigen in saline. Three to fourdays later, the animals are sacrificed, splenocytes harvested, and fusedto a murine myeloma cell line (e.g., NS1 or preferably Ag 8.653 [ATCCCRL 1580]). Hybridoma cell lines generated by this procedure are platedin multiple microtiter plates in a selective medium (for example, onecontaining hypoxanthine, aminopterin, and thymidine, or HAT) to inhibitproliferation of non-fused cells, myeloma-myeloma hybrids, andsplenocyte-splenocyte hybrids.

Hybridoma lines thus generated can be screened by ELISA for reactivitywith LbeIF4A, for example, by adaptations of the techniques disclosed byEngvall et al. (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004.A preferred screening technique is the antibody capture techniquedescribed by Beckman et al., J. Immunol. 144:4212 (1990). The hybridomalines are cloned, for example, by limiting dilution or by cloning insoft agar, to yield a monoclonal cell line. Positive clones are theninjected into the peritoneal cavities of syngeneic rodents to produceascites containing high concentrations (>1 mg/ml) of anti-LbeIF4Amonoclonal antibody. The resulting monoclonal antibody can be purifiedby ammonium sulfate precipitation followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can also be used, as canaffinity chromatography based upon binding to LbeIF4A.

Example 5 LbeIF4A Stimulation of PBMC Proliferation

This example illustrates the ability of purified recombinant LbeIF4A tostimulate proliferation of PBMCs from L. braziliensis-infectedindividuals. Peripheral blood was obtained from individuals living in anarea (Corte de Pedra, Bahia, Brazil) endemic to L. braziliensistransmission where epidemiological, clinical, and immunological studieshave been performed for over a decade. Diagnosis of the patients wasmade by clinical findings associated with at least one of the following:isolation of parasite from lesions, a positive skin test with Leishmanialysate or a positive serological test.

Peripheral blood was collected and PBMCs isolated by densitycentrifugation through Ficoll™ (Winthrop Laboratories, New York). For invitro proliferation assays, 2-4×10⁵ cells/well were cultured in completemedium (RPMI 1640 supplemented with gentamycin, 2-ME, L-glutamine, and10% screened pooled A+ human serum; Trimar, Hollywood, Calif.) in96-well flat bottom plates with or without 10 μg/ml of the indicatedantigens or 5 μg/ml PHA (Sigma Immunochemicals, St. Louis, Mo.) for fivedays. The cells were then pulsed with 1 μCi of [³H] thymidine for thefinal 18 hours of culture.

Data are represented as mean cpm of triplicate cultures and thestimulation index (SI) defined as mean cpm of cultures withantigens/mean cpm of cultures without antigen. As shown in Table I andFIG. 3, PBMCs from most (>70%) mucosal and active or healed cutaneouspatients responded to LbeIF4A with a heterogeneous proliferation patternwith stimulation indices to ranging from 12 to 233 and 2 to 64respectively.

TABLE I In Vitro Proliferation of PBMCs from L. braziliensis-infectedIndividuals in Response to Parasite Lysate and LbeIF4A Antigens [³H]TdrIncorporation (Mean cpm (SD) × 10⁻³) PATIENTS MEDIA LYSATE S.I. LbeIF4AS.I. MUCOSAL JV 0.15 (0.0) 41.30 (1.3) 294 11.90 (4.8) 81 SZ 0.45 (0.1)140.60 (7.6) 308 105.90 (5.6) 233 AB 0.42 (0.3) 44.20 (0.5) 104 5.00(1.3) 12 NO 0.38 (0.1) 52.70 (3.3) 138 12.80 (1.6) 33 TE 0.18 (0.0)27.40 (1.5) 150 8.80 (0.3) 48 MB 0.18 (0.0) 300.10 (9.4) 1634 41.50(4.5) 226.1 OM 0.28 (0.0) 35.40 (3.2) 124 6.90 (2.5) 24 CUTANEOUS AS0.22 (0.0) 19.14 (1.3) 87 14.30 (2.3) 64 JP 0.25 (0.0) 55.63 (8.6) 2184.40 (0.3) 17 VS 0.17 (0.0) 0.26 (0.0) 1.5 0.3 (0.0) 2 RJ 0.10 (0.0)0.32 (0.2) 3.0 1.5 (0.6) 15 JA 0.16 (0.0) 0.77 (0.1) 4.7 2.5 (0.2) 16 AD4.20 (1.0) 4.01 (1.0) 0.0 14.1 (2.2) 3.5 HN 0.36 (0.0) 4.73 (1.7) 134.69 (1.7) 13 DIFFUSE CUTANEOUS VAL 0.22 (0.0) 0.51 (0.3) 2.0 2.12 (0.2)9.0 SELF-HEALING CUTANEOUS GS 0.21 (0.0) 19.70 (4.4) 94 41.50 (2.8) 198MS 0.09 (0.0) 0.60 (0.1) 6.5 5.10 (2.1) 57 AH 0.11 (0.0) 59.60 (7.1) 5199.60 (4.7) 83 DJ 0.12 (0.0) 0.20 (0.1) 1.6 19.00 (6.7) 151 HS 0.12 (0.0)27.10 (2.0) 225 12.40 (2.7) 103 MCT 0.38 (0.0) 130.30 (14) 340 6.20(1.5) NORMAL LV 0.14 (0.0) 0.19 (0.0) 1.4 0.71 (0.1) 4.0 VV 0.18 (0.0)0.31 (0.1) 1.7 0.28 (0.1) 1.5 N3 0.14 (0.0) 0.36 (0.1) 2.6 0.27 (0.1)1.9 N4 0.59 (0.1) 2.00 (0.3) 3.8 0.56 (0.0) 1.0

In general, the stimulation indices were higher with PBMCs from mucosalindividuals. PBMCs from some mucosal patients responded to LbeIF4A withstimulation indices comparable to those observed with parasite lysate.Interestingly, in some patients with cutaneous leishmaniasis, theproliferative responses to LbeIF4A were higher than those elicited byparasite lysate. In contrast to mucosal and cutaneous patients, PBMCsfrom all six individuals with self healing cutaneous leishmaniasisproliferated in response to LbeIF4A with stimulation indices (16-198)comparable to those of mucosal individuals. PBMCs from two of the selfhealing individuals (MS and DJ), had responses that were significantlyhigher than those obtained with parasite lysate. Cells from normaluninfected individuals were only marginally stimulated by LbeIF4A.

Example 6 LbeIF4A Stimulation of Cytokine mRNA Expression in PBMCs

This example presents an analysis of cytokine mRNA expression patternsof PBMCs from patients with confirmed cases of L. braziliensisinfection. For cytokine mRNA analysis, 0.5 to 1 ml of PBMCs werecultured at 1-2×10⁶ cells/ml with or without 10 μg/ml of the LbeIF4Aantigen lacking the N-terminal 48 residues of SEQ ID NO:2 (as describedin Example 3) for 48 and 72 hours. The supernatants and cells wereharvested and analyzed for cytokine mRNAs by polymerase chain reaction(PCR). For cytokine mRNA PCR analysis, total RNA was isolated from thePBMCs using the acid guanidium thiocyanate-phenol-chloroform extractionmethod, as described by Chomczynski and Sacchi (Anal. Biochem.162:156-159, 1987). Complementary DNA (cDNA) was synthesized usingpoly(dT) (Pharmacia) and AMV reverse transcriptase (Bethesda ResearchLaboratories, Gaithersburg, Md.) in a final volume of 20 μl. cDNAsamples were brought to 200 μl with water.

Following normalization to β-actin, 12 to 20 μl of diluted cDNA wereamplified by PCR using Taq polymerase (Perkin-Elmer Cetus, Norwalk,Conn.) with 0.2 μM of the respective 5′ and 3′ external primers in areaction volume of 50 μl. The conditions used were: denaturation at 94°C. (1 minute for β-actin, IL-2, and IL-4; 45 sec for IFN-γ and 30 secfor IL-10), annealing at 55° C. (1 minute for β-actin, IL-2, and IL-4;30 sec for IL-10) or 60° C. for 45 sec for IFN-γ and elongation at 72°C. We verified that our PCR conditions were within the semi-quantitativerange by initially performing serial dilutions of the cDNAs and varyingthe number of cycles used for PCR. In all subsequent experiments, 30cycles were used in the amplification reactions for β-actin, IL-2, IL-4,and IFN-γ. In the case of IL-10 PCR, 25 cycles were used.

The primer pairs used and the PCR conditions were from publishedinformation; β-actin, IL-2, IL-4 and IFN-γ (Ehlers et al., J. Exp. Med.173:23-36, 1991) and IL-10 (Viera et al., Proc. Natl. Acad. Sci. U.S.A.88:1172-1176, 1991). The nucleotide sequences for the 5′ and 3′oligonucleotide primers, respectively, were as follows: (1) β-actin,TGACGGGGTCACCCACACTGTGCCCATCTA and CTAGAAGCATTGCGGTGGACGATGGAGGG; (2)IL-2, ATGTACAGGATGCA ACTCCTGTCTT and GTCAGTGTTGAGATGATGCTTTGAC; (3)IL-4, ATGGGTCTCACCTCCCAACTGCT and CGAACACTTTGAATATTTCTCT CTCAT; (4)IFN-γ, ATGAAATATACAAGTTATATCTTGGCTTT and GATGCTCTTCGACCTCGAAACAGCAT; (5)IL-10, TCTCAAGGGGCTGG GTCAGCTATCCCA and ATGCCCCAAGCTGAGAACCAAGACCCA.

Probes were obtained using plasmids containing the human sequences IL-2,IFN-γ and IL-4 (Lewis et al., Proc. Natl. Acad. Sci. U.S.A.85:9743-9747, 1988) and β-actin (no. 65128; American Type CultureCollection, Rockville, Md.), which were digested with HindIII/EcoRI,EcoRI, SacI/HindIII, and EcoRI respectively. Human IL-10 cDNA was clonedby PCR from mitogen-stimulated PBMCs from normal donors usingoligonucleotide primers designed to amplify a 535 base pair fragmentspanning the entire coding region of human IL-10 (Lewis et al., Proc.Natl. Acad. Sci. U.S.A. 85:9743-9747, 1988). The cDNA was subcloned intopBluescript and digested with BamHI/EcoRI. After separation on 1%agarose gels, insert DNA fragments were excised, electroeluted, andpurified. Radiolabeled ³²P-probes were prepared by the random primingmethod.

PCR products were analyzed by electrophoresis on 1.5% agarose gels,transferred to nylon membranes, and probed with the appropriate³²P-labeled DNA insert. Hybridizations were at 55° C. overnight. Posthybridization washes were at 55° C. for 20 minutes twice each with 2×,and 1×SSC containing 0.2% SDS.

The results of these analyses are presented in FIGS. 4A and 4B. PCRcytokine analyses were performed with cells prior to culturing (lanes0), following culturing in the absence of antigen (lanes −), orfollowing culturing in the presence of 10 μg/ml L. braziliensis lysate(lanes Lb) or in the presence of 10 μg/ml LbeIF4A (lanes IF). FIG. 4Ashows the PCR results of cytokine mRNA for three of the six mucosalpatients' PBMCs analyzed (JV, SZ, and TE) and one patient (VA) with L.amazonensis infection, manifested as diffuse cutaneous leishmaniasis(DCL). In three of the six mucosal patients (TE, FIG. 4A; NO and EO, notshown), PBMCs not cultured in vitro had detectable levels of mRNA forIFN-γ and IL-4, as well as IL-2 (patients TE and EO). IL-10 mRNA was notdetected in the “resting” PBMCs from any of the mucosal patients.However, following in vitro culturing in the absence of antigenstimulation, the synthesis of IL-10 MRNA was upregulated in most of themucosal PBMCs analyzed. In addition, the levels of cytokine mRNAsdetected in the “resting” PBMCs of patients TE, NO, and EO, decreased tobackground levels.

Parasite lysate stimulated the expression of mRNAs of the Th1 cytokinesIFN-γ and IL-2 as well as that of the Th2 cytokine IL-4 (in three of thesix patients). Increased IL-10 mRNA was detected in one of the patients'PBMCs (SZ) following culture with the parasite lysate. Both LbeIF4Aantigen and parasite lysate elicited the production of mRNA of IFN-γ andIL-2 from all mucosal patient PBMCs with LbeIF4A eliciting an exclusiveTh1 cytokine profile. In fact, LbeIF4A downregulated the synthesis ofIL-10 mRNA detected in the cultured PBMCs of most mucosal patients priorto antigen stimulation. Interestingly, as with the case of using PBMCsfrom mucosal patients, LbeIF4A also downregulated the synthesis of IL-10mRNA in the DCL patient VA.

In general, the levels of mRNAs for IFN-γ and IL-2 increased fromundetectable amounts prior to antigen stimulation to readily visuallevels following antigen stimulation in ethidium bromide stained gels.However, mRNA for the cytokines IL-4 and IL-10, were only detectedfollowing radioactive probing of the resolved PCR products, indicatinglow abundance of these cytokine messages.

Similar PCR analysis was performed on PBMCs derived from cutaneouspatients (FIG. 4B). The fresh PBMCs from three (VS, JP and CA (notshown)) of the four patients analyzed revealed high levels of mRNAs forboth the Th1 (IFN-γ and (IL-2) and Th2 (IL-4 and IL-10) cytokinesexamined. mRNAs for IFN-γ and IL-2, but not for IL-10 and IL-4, weredetected in the fresh PBMCs of the fourth (AS) cutaneous patient.Therefore, in contrast to mucosal patients, patients with cutaneousleishmaniasis have IL-10 mRNA, in addition to IL-4, IL-2, and IFN-γ, intheir fresh PBMCs. Interestingly, while the mRNAs for IL-2 and IFN-γwere reduced to barely detectable levels following the in vitroculturing of PBMCs in the absence of antigen, those for IL-10 remainedeither unaffected or increased. Therefore, in cutaneous patients, thespontaneous levels of IL-10 mRNA is either stable or their PBMCscontinue to synthesize IL-10 mRNA in the absence of antigen stimulation.The observation of such a response for cutaneous leishmaniasis patientscan be exploited to differentiate individuals who are predisposed todeveloping chronic cutaneous leishmaniasis from those who willexperience self healing lesions.

All cutaneous patients tested responded to LbeIF4A antigen as well as tothe parasite lysate by upregulating the synthesis of mRNAs for IL-2 andIFN-γ and, in two of four patients (VS and AS), the level of IL-4 mRNAalso increased following stimulation with parasite lysate. In the threepatients (VS, JP and CA) with detectable spontaneous levels of IL-10mRNA, LbeIF4A as well as the parasite lysate down-regulated theexpression of IL-10, mRNA.

The cytokine mRNA profiles of PBMCs from patients with self-healing CLwere similar to those of ML patients in that (a) except for oneindividual with detectable levels of IL-10 mRNA, fresh PBMCs from threeof four patients analyzed had detectable levels of IL-2, IFN-γ and IL-4,but little or no IL-10 mRNA; (b) IL-10 mRNA was upregulated afterculture of PBMCs without antigen, whereas those of IL-2, IFN-γ and IL-4decreased to background levels; and (c) leishmanial lysate stimulatedthe expression of a mixed Th1/Th2 cytokine profile, whereas LbeIF4Aelicited increased mRNA expression of only the Th1-type cytokines anddownregulated the expression of IL-10 mRNA in the cultured PBMCs of mostself-healing individuals (not shown).

Example 7 LbeIF4A Stimulation of Cytokine Secretion in PBMCs

This example presents the supernatant levels of secreted cytokines ofPBMCs from L. braziliensis-infected individuals following stimulationwith LbeIF4A antigen lacking the N-terminal 48 residues of SEQ ID NO:2(as described in Example 3) or parasite lysate. Aliquots of the PBMCsupernatants were assayed for IFN-γ, TNF-α, IL-4, and IL-10. IFN-γ wasquantitated by a double sandwich ELISA using mouse anti-human IFN-γ mAb(Chemicon, Temucula, Calif.) and polyclonal rabbit anti-human IFN-γserum. Human rIFN-γ (Genentech Inc., San Francisco, Calif.) was used togenerate a standard curve. IL-4 was quantitated in supernatants by adouble sandwich ELISA using a mouse anti-human IL-4 mAb (M1) and apolyclonal rabbit anti-human IL-4 sera (P3). Human IL-4 (Immunex Corp.Seattle, Wash.) was used to generate a standard curve ranging from 50pg/ml to 1 ng/ml. IL-10 was measured using a rat anti-human IL-10 mAb(PharMingen, San Diego, Calif., Cat. #18551D) to “capture” secretedIL-10 and a biotinylated rat antihuman IL-10 mAb (PharMingen San Diego,Calif., Cat. #18562D) for detection of bound IL-10 with streptavidinconjugated horse radish peroxidase and ABTS as substrate. A standardcurve was obtained using human rIL-10 (kindly provided by DNAX ResearchInstitute, Palo Alto, Calif.), ranging from 30 pg to 2 ng/ml.

Cells from all three patient groups (i.e., mucosal, cutaneous andself-healing cutaneous) secreted IFN-γ and TNF-α following stimulationwith either 10 μg/ml LbeIF4A antigen or 10 μg/ml parasite lysate (FIGS.5 and 6). Similarly, LbeIF4A stimulated PBMCs from patients with L.tropica infection (Desert Storm Patients) to proliferate and secreteIFN-γ (not shown). The levels of both IFN-γ and TNF-α detected in thesupernatants of patient PBMCs were significantly higher than those fromuninfected controls. In the absence of antigen stimulation, only PBMCsfrom mucosal patients (five of six) produced detectable levels ofsupernatant TNF-α (60 to 190 pg/ml). Little or no IL-4 or IL-10 wasdetected in any of the supernatants analyzed (not shown), indicatinglevels below the detection limit of the ELISA assay employed. Bycomparison, leishmanial lysate also stimulated PBMCs to secrete IFN-γand TNF-α and, in some patients. IL-10 was also detected (not shown).Taken together, the results demonstrate that LbeIF4A stimulated apredominant Th1 cytokine profile in PBMCs from L. braziliensis-infectedindividuals, whereas parasite lysate stimulated a mixed Th1/Th2 cytokineprofile.

The levels of TNF-α detected in the supernatants of patient PBMCs frommucosal and self-healing individuals following antigen stimulation werehigher than those from cutaneous patients (FIG. 6). PBMCs from four offive mucosal patients (JV, SZ, AB, and MB) had supernatant levels ofTNF-α (0.80 to 2.20 ng/ml) higher than those detected in cultures ofPBMCs from uninfected controls following stimulation with parasitelysate. Similarly, the same PBMCs were stimulated by LbeIF4A to producesupernatant levels of TNF-α with values ranging from 0.66 to 3.14 ng/ml.Compared to uninfected controls, PBMCs from three (GS, HS, and MCT) outof six self-healing individuals analyzed produced higher levels of TNF-αin response to parasite lysate, and all six (GS, MS, AH, DJ, HS, andMCT) out of six self-healing individuals analyzed produced higher levelsof TNF-α in response to LbeIF4A. The levels of TNF-α produced by PBMCsfrom cutaneous leishmaniasis patients in response to parasite lysatewere comparable to uninfected controls. However, LbeIF4A stimulatedPBMCs in three of these patients (RJ, AD and JS) to produce TNF-α. Suchpatients may be in the process of developing acute cutaneousleishmaniasis.

Example 8 Stimulation of IL-12 Production by LbeIF4A

This example shows that LbeIF4A stimulates PBMCs from L.braziliensis-infected individuals, as well as PBMCs or cultured humanmacrophages, adherent PBMCs, from the blood of normal donors and thehuman myeloid leukemia cell-line THP-1, to secrete IL-12. IL-12 has beenshown to play a pivotal immunoregulatory role in the development of cellmediated immunity, generation of Th1 responses and IFN-γ production inintracellular bacterial or parasitic infections. The LbeIF4A polypeptideused was the LbeIF4A antigen lacking the N-terminal 48 residues of SEQID NO:2 (as described in Example 3).

IL-12 p40 was measured in cell-free supernatants by RIA (detection limitof 10 pg/ml) using the mAb pairs C11.79/C8.6, as described by D'Andreaet al. (J. Exp. Med. 179:1387-1398, 1992). Biologically active IL-12 p70heterodimer (detection limit 1 pg/ml) was measured as described by Kubinet al. (Blood 83:1847-1855, 1994).

FIG. 7A shows that 10 μg/ml LbeIF4A (LeIF) stimulated mucosal patientPBMCs to secrete IL-12 p40 in the cultured supernatant with a magnitudesignificantly higher than the IL-12 p40 levels observed with 10 μg/mlparasite lysate as antigen (Lb). The amount of IL-12 p40 secreted in theabsence of lysate or antigen is also shown (Med). The same figure alsoshows that 10 μg/ml IL-10 down-regulated the production of IL-12 p40 bypatient PBMCs following stimulation with LbeIF4A (LeIF+IL-10) or lysate(Lb+IL-10).

PBMCs from uninfected individuals also produced IL-12 p40 when culturedwith LbeIF4A (LeIF, FIG. 7B), although no p40 was detected in responseto parasite lysate (Lb). This may suggest a role for IFN-γ in thelysate-induced p40 observed in patient PBMCs, which produced 5-100 foldmore IFN-γ than normal PBMCs after antigen stimulation (see FIG. 5).

To determine whether the IL-12 p40 observed in antigen-stimulated PBMCcultures reflected biologically active cytokine, IL-12 p70 was alsoassayed in these cultures (FIGS. 7C and 7D). In general, the p70production paralleled that of p40, demonstrating that biologicallyactive IL-12 was produced in response to LbeIF4A in both patient andnormal PBMCs.

LbeIF4A also stimulates IL-12 production in cultured human macrophages(FIG. 9A) and in adherent PBMCs (FIG. 9B). Adherent cells were preparedfrom PBMCs separated by Ficoll-hypaque gradient centrifugation from theblood of normal donors. 2×10⁶ PBMCs were cultivated for 2 hours in 500μl RPMI, 2% human AB serum. Adherent cells were purified by washing theplates 3 times with PBS. Then 500 μl of test medium (RPMI, 2% human ABserum) with the respective stimulus were added (IFN-1000 U/ml, LbeIF4A(Lf) 10 μg/ml). Supernatants were taken after 18 hours.

IL-12 production of adherent PBMCs was measured by a capture bio-assaywith 5 day old PHA blast. Briefly, the IL-12 capture antibody C11.5.14(kind gift of the Wistar Institute) was coated on 96 well plates.Supernatants of the induction experiment and recombinant IL-12, as astandard, were incubated for 4 hours. After several wash steps, 5 dayold PHA blasts were added and the proliferation of these blasts was usedto determine IL-12 concentrations in supernatants of adherent cells.

Macrophages were generated by cultivating adherent cells (2×10⁶ PBMCs)for 5 days in test medium. Then, the macrophages were washed in PBS and500 μl RPMI, 2% human AB serum, and 1000 U/mL IFN-γ was added.Macrophages were stimulated with LbeIF4A (10 μg/ml) or cultivated inmedium (M) alone. In one set, LbeIF4A control macrophages were incubatedwith LbeIF4A in 500 μl RPMI, 2% human AB serum, without IFN-γ.Supernatants were taken after 18 hours and used for induction of IL-12dependent proliferation. Briefly, 5 day old blasts were incubated withmacrophage supernatants for 2 days. For the last 18 hours, ³H thymidinewas added. Neutralizing anti-IL-12 polyclonal goat serum (5 μg/ml) wasadded as indicated.

In addition, LbeIF4A stimulates IL-12 p40 production in the humanmyeloid leukemia cell-line. THP-1 (FIG. 10). The cells were cultured at106 cells/ml for 24-48 hours in Endotoxin-free RPMI medium containing 5%Fetal Bovine serum. 10 μg/ml LbeIF4A synergized with IFN-γ to stimulateTHP-1 cells to secrete IL-12 p40. These results indicate the utility ofLbeIF4A as vaccine adjuvant.

Example 9 Effect of IL-12 and IL-10 on LbeIF4A Induction of IFN-γProduction

This Example examines the interaction among IL-12, IL-10 and IFN-γ inresponse to the LbeIF4A polypeptide lacking the N-terminal 48 residuesof SEQ ID NO:2 (as described in Example 3). As shown in FIG. 8A, PBMCsfrom patients with mucosal leishmaniasis were stimulated with 10 μg/mlLbeIF4A in the absence (LeIF) or presence of 10 ng/ml anti-IL-12(LeIF+Anti-IL-12), or IL-10 (LeIF+IL-10), and the cultured supernatantswere assayed for IFN-γ secretion. Both anti-IL-12 mAb and IL-10abrogated the production of LbeIF4A-induced IFN-γ secretion. However,anti-IL-12 mAb only partially decreased the production of IFN-γ afterstimulation with leishmanial lysate (FIG. 8B). These results show thatIFN-γ production is IL-12 dependent, and is inhibited by IL-10, whereasthe production of IL-12 is regulated by both IFN-γ dependent andindependent pathways.

Example 10 LbeIF4A Stimulation of a TH1 Profile in Mice

This example demonstrates that the LbeIF4A polypeptide lacking theN-terminal 48 residues of SEQ ID NO:2 (as described in Example 3)stimulates a dominant TH1 cytokine profile in BALB/c mice. The animalswere primed with either LbeIF4A or 8E (the C-terminal portion of the L.braziliensis mitochondrial hsp70, which stimulates patient PBMCs toproduce high levels of IL-10) using quilA or CFA as adjuvants. Ten daysafter priming, lymph node (LN) cells were restimulated in vitro with therecombinant antigens and the supernatant cultures were analyzed forsecreted cytokines. The results (FIG. 1 1) show that LN cells of miceprimed with LbeIF4A proliferated and secreted an almost exclusive Th1cytokine (IFN-γ) following challenge with LbeIF4A using both types ofadjuvants. In contrast, LN cells from mice primed with 8E produced a Th0response or Th1/Th2 type cytokine (with quilA as adjuvant) with a strongbias towards the Th2 cytokines, IL-4, and IL-10 in specific response tochallenge with 8E. Similarly, mice primed with parasite lysate produceda mixed cytokine profile, a result that may argue against the use ofparasite lysate alone as vaccine candidate (FIG. 11).

These results indicate that LbeIF4A may be used as an adjuvant, as wellas a specific T cell vaccine. Because LbeIF4A induced a powerful Th1response, including the two cytokines most clearly associated withprotection in experimental leishmaniasis, IFN-γ and IL-12, we studiedthe ability of this antigen to protect mice against leishmaniasis.BALB/c mice were immunized once with LbeIF4A with no adjuvant, followedby subcutaneous infection with L. major seven days later. Compared tothe control group, LbeIF4A provided significant protection against L.major infection (FIG. 12). Thus a heterologous antigen derived from L.braziliensis can confer some protection to L. major infection,suggesting that, at least some of the “protective” epitopes areconserved between the two parasites.

Example 11 Preparation of LmeIF4A

This example illustrates the appropriation of new variant of LbeIF4Afrom Leishmania major.

A cDNA expression library was constructed with polyA⁺ RNA of L. majorusing the ZAP-cDNA unidirectional cloning kit (Stratagene, La Jolla,Calif.). Approximately 500,000 pfu was screened with a radio-labeled DNAprobe comprising nucleotides 258 to 1188 of SEQ ID NO. 1.Post-hybridization washes were at 55° C. with 0.5×SSC containing 0.1%SDS. This resulted in the identification of a clone containing anapproximately 2.5 kb insert (LmeIF4A). Excision of the pBSK (−) phagemidsequences was carried out according to the manufacturer's protocols.Overlapping clones were generated from the cDNA insert by exonucleaseIII and sequenced by the Tag dye terminator.

The approximately 2.7 kb cDNA insert of LmeIF4A was found to contain theentire coding sequence, as well as the 5′ spliced leader and 3′ flankingsequences. A fragment containing the entire coding sequence of LmeIF4Awas amplified by PCR using 5′ and 3′ specific oligonucleotides. The 5′oligonucleotide contained an NdeI restriction site preceding the ATGinitiation site. When cloned into the NdeI site of the pET vector(Novagen, Madison, Wis.), the first amino acid of the protein was theinitiation code (Met) of the non-fusion recombinant LmeIF4A. Non-fusionfull-length rLmeIF4A was produced in E. coli, using the pET plasmavector and a T7 polymerase expression system (Novagen, Madison, Wis.).The inclusion bodies were isolated and sequentially washed twice in 10ml of TNE (50 mM TRIS, pH 8.0, 100 mM NaCl, 10 mM EDTA) containing 2, 4,and 8 M urea. The 8 M urea fraction containing a recombinant antigen wasapplied to a preparative SDS-PAGE gel. Recombinant LmeIF4A was purifiedeither by electroelution from the preparative gel or by HPLC. The finalproduct had negligible levels of bacterial endotoxin, as determined byLimulus assay.

Partial sequence analysis of the LmeIF4A cDNA indicated substantialhomology (at the nucleotide and amino acid level) (SEQ ID NOS:3 and 4)to the antigen isolated from L. braziliensis (i.e., greater than 90%amino acid sequence identity).

Example 12 Elicitation of Antigen-Specific CTL Response

This example illustrates the elicitation of a specific CTL responseagainst soluble ovalbumin using LmeIF4A, prepared as described above.

C57BL/6 mice (H-2^(b)) were immunized once with 30 μg of solubleovalbumin (Sigma, St. Louis, Mo.), either alone (ova alone) or incombination with 50 μg of LmeIF4A encapsulated in poly-lactide-glycolidebeads (Southern Research Institute, Birmingham, Ala.) (Lmeif/PLG).Groups of mice were also immunized with LmeIF4A encapsulated in PLGbeads (ova plus Lmeif/PLG) or PLG alone. The size of beads used for theexperiment was in the range of 1 to 10 μm.

Three weeks after immunization, mice were sacrificed and the spleensremoved. To generate effector cells, spleen cells were prepared andstimulated in vitro with the ova expressing EG7.ova cells, afterirradiation (20.000 rad) for five days. The EG7.ova cell line wasgenerated by transfecting the EL4 thymoma cell line (H-2^(b))(Infectious Disease Research Institute, Seattle. Wash.) with the ovacDNA-containing plasmid under the control of β-actin as described (Mooreet al., Cell 54:777-785 (1988)).

Priming was assessed by the presence of effectors capable of lysing ⁵¹Crlabeled EG7.ova, without lysing ⁵¹Cr labeled non-transfectant parentalEL4 cells. The results are shown in FIG. 13, and are expressed as %cytotoxicity as a function of effector to target cell ratio. Theseresults demonstrate that mice immunized with soluble ovalbumin incombination with LmeIF4A show an excellent CTL response which killedtarget cells specifically. No specific CTL elicitation could be detectedin mice immunized with either LmeIF4A, PLG or soluble ovalbumin alone.

Example 13 Stimulation of Trinitrophenol-specific Antibodies

This example illustrates the enhancement of anti-trinitrophenol (TNP)antibodies using LmeIF4A.

C57BL/6 mice were immunized (day 0) intraperitoneally with 3 μg TNP-KLHcombined with either alum, LmeIF4A (75 μg) or with saline. The mice werebled on day 10. boosted (same as primary) on day 21 and bled on day 26.The sera were tested for TNP specific, isotype specific antibodyresponses by enzyme-linked immunosorbant assay (ELISA). Data areexpressed, in FIG. 14, as the log of the dilution of each antiserum thatgave a 50 percent of maximum OD in each ELISA, as calculated by linearregression analysis.

These results show a significant enhancement of antibody response byLmeIF4A, as compared to immunization with antigen alone. Of particularinterest, no noticeable increase in IgE, the antibody class mostassociated with the Th2 response, was found. In addition, primary IgMand IgG antibody responses were enhanced after a single injection ofTNP-KLH (3 μg) with LmeIF4A (75 μg), as described above.

Example 14 Enhancement of Production of MUC-1 Specific Antibodies

This example illustrates the use of LmeIF4A to enhance the production ofMUC-1 specific antibodies.

C57BL/6 mice were immunized with human tumor antigen MUC-1 in variouscombinations with LmeIF4A. Groups included mice immunized with solubleMUC-1 with or without soluble LmeIF4A and MUC-1 immobilized on PLG beadswith or without soluble LmeIF4A. For MUC-1/PLG experiments, two separatebead sizes were used. One batch of beads were used for 1-10 μm in sizewhile the other ranged from 40-100 μm. Ten, twenty and thirty days afterimmunization, mice were bled and the sera were tested for the presenceof MUC-1 specific antibodies (IgG and IgM). The sera were also testedfor the presence of μ, γ2a, γ2b and γ1 isotypes.

The results, shown in FIG. 15, demonstrate that, while one immunizationwith the MUC-1 or MUC-1 plus LmeIF4A was not sufficient to generate ananti-MUC-1 antibody response, LmeIF4A significantly enhanced theantibody response when MUC-1 was encapsulated in PLG beads. MUC-1 insmall PLG beads induced strong anti-MUC-1 responses deductible as earlyas 10 days after the first immunization and the addition of LmeIF4A tothe preparation further enhanced the antibody response. One immunizationwith MUC-1 in large PLG beads did not produce any detectable anti-MUC-1antibodies within 10 days of immunization, but an amplified response wasobserved in mice immunized with MUC-1 in large PLG beads in combinationwith LmeIF4A. The isotope distribution of anti-MUC-1 antibodiesconsisted of IgM, IgGγ2a, IgGγ2b, and IgGγ1 with no IgE responsedetected.

Example 15 Enhancement of CTL Activity in Cultured Cells

This example illustrates enhancement of specific CTL activity by LmeIF4Ain cultured cells.

Mice were immunized with 30 μg of soluble ovalbumin or the sameconcentration of ovalbumin encapsulated in PLG beads. After two weeks,spleen cells were removed from immunized mice and stimulated in vitrowith no antigen, LmeIF4A alone (10 μg/ml) irradiated EG7.ova orirradiated EG7.ova plus LmeIF4A at the same concentration. The resultsare shown in FIG. 16.

These results indicate that ovalbumin/PLG immunized mice can primeovalbumin-specific CTL in vivo which can be detected by stimulatingresponder spleen cells with EG7.ova stimulator cells. Furthermore, CTLgeneration from ovalbumin/PLG primed spleen cells can be substantiallyaugmented by the addition of LmeIF4A (10 μg/ml) to the EG7.ovacontaining cultures. It should be noted that in the first in vitroculture with EG7.ova or EG7.ova plus LmeIF4A, total mononuclear cellcounts did not differ (see Tables 2 and 3 below).

TABLE II LmeIF4A Enhances CTL Activities Ex Vivo Lytic Units (LU)/10⁶Immunized With Stimulated ex vivo with Mononuclear Cells None No antigen0 LmeIF4A alone 0 EG7.Ova 0 EG7.Ova plus LmeIF4A 0 Bead alone No antigen0 LmeIF4A alone 0 EG7.Ova 0 EG7.Ova plus LmeIF4A 0 Ova/bead No antigen 0LmeIF4A alone 0 EG7.Ova 2.5 EG7.Ova plus LmeIF4A 16.6

TABLE III Recovery of Mononuclear Cells from LmeIF4A Containing CulturesNo. of Cells (LU)/10⁶ Immunized Recovered/10⁷ With In Vitro StimulatedWith Mononuclear Cells Added Ova/PLGA No Antigen 1.8 LmeIF4A alone 2.4EG7.Ova 2.52 EG7.Ova plus LmeIF4A 2.88 Soluble Ova 3.2 Soluble Ova plusLmeIF4A 2.6

We also evaluated whether LmeIF4A can be used to potentiate and expandantigen specific CTL activities in vitro. CTLs generated fromovalbumin/PLG-immunized mice discussed above were restimulated in vitrowith either irradiated EG7.ova plus IL-2 or with EG7.ova plus IL-2 andLmeIF4A. After five days of culture, effector cells were tested against⁵¹Cr-labelled EL4 cells or EG7.ova cells.

The results, shown in FIG. 17, indicate that ovalbumin-specific CTLrestimulated with EG7.ova plus LmeIF4A and IL-2 killed EG7.ova betterthan the same effectors generated with EG7.ova plus IL-2 alone, but thedifference does not appear to be significant. However, when the cellcounts were taken, the culture with LmeIF4A produced 2.5-fold highercells than the cultures without LmeIF4A. These results, shown below inTable 4, are expressed in lytic units per culture, and demonstrateoverall strength of LmeIF4A to expand specific CTL numbers in vitro.

TABLE IV Recovery of Mononuclear Cells from LmeIF4A Containing CulturesNo. of Cells (10⁶) Recovered/10⁶ Total Immunized In Vitro MononuclearLytic Units With Stimulated With Cells Added (LU50)/Culture Ova/PLGA smEG7.Ova plus IL-2 6.4 75.2 Nr EG7.Ova plus IL-2 16 388.8 and LmeIF4A

Example 16 Augmentation of the Induction of Alloreactive CTL by LmeIF4A

This examples illustrates the induction of murine alloreactive CTL byLmeIF4A.

2.5×10⁶ BALB/c spleen cells were cultured with 5×10⁶ irradiated (3500 R)C57BL/6 spleen cells in 2 ml of RPMI:FCS in the presence or absence of10 μg/ml gel-purified LmeIF4A. Cultures were harvested on day 5, washedand tested at different effector cell concentrations (culture fraction)for cytolytic activity against ⁵¹Cr labelled EL4 cells (2,000/well) in a4 hr release assay in 200 μl cultures. Data are expressed, in FIG. 18,as percent specific release, which is calculated as:

100×(CPM experimental)−(CPM spontaneous)/(CPM maximum)−(CPM spontaneous)

These results indicate that LmeIF4A is capable of significantlyenhancing alloreactive CTL. Taken together with the other resultsdescribed above, the Leishmania eIF4A homologue appears to have potentadjuvant activity in a variety of assay systems. Furthermore, itsability to induce IL-12, together with its lack of toxicity, makes it aunique adjuvant.

Example 17 Tumor Regression After Administration of LmeIF4A and Antigen

Female C57BL/6 (H-2^(b)) mice of 6-8 weeks of age were immunized oncewith a subcutaneous injection with 50 μg LmeIF4A either alone or 30 μgencapsulated in poly-lactide-glycolide beads (LbeIF/PLG), VSV peptidesalone or in PLG beads, GM-CSF in PLG beads, or vehicle (PBS). N1 cellswere generated from the EL4 cell by transfection of the VSV (vesicularstomatitis virus) nucleocapsid protein gene. Because the plasmiddirecting expression of the VSV nucleocapsid protein contains theneomycin resistance gene as a selectable marker, the cell line wasmaintained in a selective medium containing G418. Recombinant LmeIF4Awas produced from E. coli transfectants and purified by HPLCfractionation. N1-specific peptide antigen was chemically synthesizedand also purified by HPLC. Proteins were encapsulated into PLGmicrospheres by Southern Research Institute (Birmingham, Ala.).

Approximately 2×10⁵ N1 cells were inoculated intradermally into theright flank of C57BL/6 mice. After palpable tumors were established,mice were randomized into groups of five and given subcutaneousinjections of the various peptides listed above on the opposite flank.Tumor growth was monitored by measuring the diameters of tumors every 2or 3 days and converting the measurement into volume according to theformula V=4/3 πr³.

The efficacy of LmeIF4A in eliciting immune responses against tumorspecific antigens was tested. In the murine tumor N1, the octamericantigenic peptide epitope RGYVYQGL is constitutively presented by theH-2K^(b) molecule. As shown in FIG. 19, the injected combination ofVSV/PLG and LmeIF4A/PLG resulted in significant suppression of tumorgrowth. Regression was not observed in the control mice or in micereceiving other combinations of antigens. A second study (FIG. 20)confirmed this result and also demonstrated that LmeIF4A is a muchbetter adjuvant than GM-CSF. In this experiment, tumors started toregress one week after antigen injection in mice administered with thetumor specific VSV/PLG and LmeIF4A/PLG. Indeed, three out of five micecompletely rejected their tumors in this particular study. In addition,soluble LeIF also had potent anti-tumor activity in combination with PLGencapsulated VSV peptide (FIG. 21).

In addition, the immune response generated through LmeIF4A and tumorantigen co-immunization was highly specific. The residual tumor mass inmice who did not completely reject N1 tumor was surgically removed. Whenthese tumor cells were cultured in medium containing 0.2 mg/ml G418,they were killed two days later. In contrast, original N1 cells used inthe tumor inoculum were resistant to G418. Thus, the residual tumor wasderived from N1 mutants that had lost the expression plasmid containingboth the antigenic VSV sequence and the neomycin resistance genes.Therefore, LeIF is capable of boosting specific immune response againsta predefined antigen, which, in turn, can lead to a therapeutic effectagainst tumors.

Example 18 Treatment of Established Tumors With LmeIF

Female C57BL/6 mice were injected s.c. with 2×10⁵ Lewis lung carcinomacells. By eight days following injection, tumors were detected in allmice. Mice were then divided into two groups of five mice each. On days8, 10, 13, and 16 after tumor inoculation, mice were injected s.c., at asite distant from the tumor, with 0.2 ml saline or 50 μg LmeIF in 0.2 mlsaline. Tumor growth was measured on days 8, 10, 13, and 16. As shown inthe following table, mice receiving LmeIF had substantially reducedtumor growth by day 16.

TABLE V Inhibition of Tumor Growth by LmeIF Average tumor volume in mm³Group day 8 day 10 day 13 day 16 Saline injected 5.21 12.61 41.06 283.53Lmeif injected 6.79 10.83 26.75 69.48

Example 19 Ability of LeIF to Influence Cytokine Response and GenerateLeIF Specific T Cell Clones

This Example illustrates the ability of LeIF to influence the Th1/Th2cytokine responses and the generation of LeIF specific T cell clones inthe absence of adjuvant.

A. Nature of LmeIF4A Specific T cell Responses in Leishmania-infectedand Uninfected BALB/c Mice.

Preparation of LmeIF4A. cDNA encoding LmeIF4A was prepared as describedabove. FIG. 22 shows a comparison of the predicted protein sequence ofLmeIF4A (SEQ ID NO:4) with the homologous sequences from L. braziliensis(LbeIF4A; SEQ ID NO:2) mouse (MeIF; SEQ ID NO:5) and human (HeIF; SEQ IDNO:6) showing that LmeIF4A has the highest sequence homology to L.braziliensis eIF protein with 99.8% total homology (98.3% identity, 1.5%conservative substitution). Both LmeIF4A and LbeIF4A are of identicallength with only seven amino acid residue substitutions (six beingconservatively over their entire lengths (FIG. 22). In contrast, LmeIF4Ashows ˜50% identity with the eIF proteins of mouse and human with the Nterminal half representing the most variable portion between the eIFproteins of Leishmania and those of mouse an human. In fact, it wasnecessary to introduce gaps in the sequences to allow for maximumalignment between the Leishmania proteins and the mammalian homologues.Despite these differences, all four proteins have a series of conservedmotifs arranged in identical order characteristic of the “DEAD box’family of RNA helicases. Two of these conserved sequences representspecialized versions of the A and B motifs previously described in otherATP binding proteins. The four amino sequence Asp-Glu-Ala-Asp (DEAD; SEQID NO:13) is part of the specialized version of the B motif. MotifI(Gly-Thr-Gly-Lys-Thr; SEQ ID NO:14) corresponds to the A-site of thenucleoside triphosphate (NTP)-binding motif and is found in mostnucleotide-binding proteins including ATPases, kinases, and DNA and RNAhelicases. MotifII corresponds to the B site of the NTP-binding motifwhich interacts through the invariant “D” residue with the Mg2+ moietyof Mg-ATP.

Full length and overlapping L. major rLeIF proteins were expressed in E.coli with six histidine residues at the amino-terminal portionimmediately following the initiator Met residue (N-terminal His-tag) ofthe pET plasmid vector (pET-17b) and a T7 RNA polymerase expressionsystem (Novagen, Madison, Wis.). The cDNA was amplified by PCR from theinitial cloned construct in pBSK(−) vector using specificoligonucleotides comprising 5′ and 3′ sequences. The specificoligonucleotide primers used for PCR amplification of the LeIF cDNA wereas follows: 1) Full length sequence (aminoacid residues 1 to 403: 5′[oligo 1 (SEQ ID NO:7)—CAA TTA CAT ATG CAT CAC CAT CAC CAT CAC ATG GCGCAG AAT GAT AAG ATC GCC] and 3′ [oligo 403 (SEQ ID NO:8)—CAT GGA ATT CCGCTT ACT CGC CAA GGTAGG CAG C]); 2) amino acid residues 1 to 226: 5′oligo-1 as above and 3′ [oligo 226 (SEQ ID NO:9)—CAT GGA ATT CTTA GTCGCG CAT GAA CTT CTT CGT CAG]; 3) amino acid residues 196 to 403: 5′[oligo 196 (SEQ ID NO:10)—CAA TTA CAT ATG CAT CAC CAT CAC CAT CAC TTCCGC TTC CTG CCG AAG GAC ATC and 3′ [oligo-403] as above; and 4) aminoacid residues 129 to 261: 5′ [oligo 129 (SEQ ID NO:11)—CAA TTA CAT ATGCAT CAC CAT CAC CAT CAC GAG ACC TTT GTC GGC GGC ACG CGC] and 3′ [oligo261 (SEQ ID NO:12)—CAT GGA ATT CTT ACA GGT CCA TCA GCG TGT CCA GCT T].The 5′ and 3′ oligonucleotides contain NdeI and EcoRI restrictionendonuclease sites (underlined) and primer sequences derived from LeIFsequence are indicated by italics with the ATG initiator and TAAterminator codons in bold. The PCR products were digested with NdeI andEcoRI and ligated into the poly-linker of pET-17b vector pre-digestedwith NdeI and EcoRI. E. coli strain BL21 (DE3) pLysE (Novagen) was usedfor high level expression.

The recombinant (His-Tag) antigens were purified from the insolubleinclusion body of 500 ml of IPTG induced batch cultures by affinitychromatography using the one step QIAexpress Ni-NTA Agarose matrix(QIAGEN, Chatsworth. Calif.) in the presence of 8M urea. Briefly, 20 mlof an overnight saturated culture of BL21 containing the pET constructwas added into 500 ml of 2×YT media containing 50 μg/ml ampicillin and34 μg/ml chloramphenicol, grown at 37° C. with shaking. The bacterialcultures were induced with 2 mM IPTG at an OD 560 of 0.3 and grown foran additional 3 hours (OD=1.3 to 1.9). Cells were harvested from 500 mlbatch cultures by centrifugation and resuspended in 20 ml of bindingbuffer (0.1 M sodium phosphate, pH 8.0; 10 mM Tris-HCl, pH 8.0)containing 2 mM PMSF and 20 μg/ml leupeptin. E. coli was lysed by adding15 mg of lysozyme and rocking for 30 minutes at 4° C. followingsonication (4×30 seconds), then spun at 12 k rpm for 30 minutes topellet the inclusion bodies.

The inclusion bodies were washed three times in 1% CHAPS in 10 mMTris-HCl (pH 8.0). This step greatly reduced the level of contaminatingLPS. The inclusion body was finally solubilized in 20 ml of bindingbuffer containing 8 M urea or 8M urea was added directly into thesoluble supernatant. Recombinant antigens with His-Tag residues werebatch bound to Ni-NTA agarose resin (5 ml resin per 500 ml inductions)by rocking at room temperature for 1 hour and the complex passed over acolumn. The flow through was passed twice over the same column and thecolumn washed three times with 30 ml each of wash buffer (0.1 M sodiumphosphate and 10 mM Tris-HCL, pH 6.3) also containing 8 M urea. Boundprotein was eluted with 30 ml of 100 mM immidazole in wash buffer and 5ml fractions collected. Fractions containing the recombinant antigenwere pooled, dialyzed against 10 nM Tris-HCl (pH 8.0) bound one moretime to the Ni-NTA matrix, eluted and dialyzed in 10 mM Tris-HCL (pH7.8). The yield of recombinant protein varies from 25-150 mg per literof induced bacterial culture with greater than 98% purity. Endotoxinlevels were typically <10 EU/mg protein (i.e., <1 ng LPS/mg).

The sizes of the expressed proteins (FIG. 23) correlated well with theirpredicted molecular weights. The yield of purified rLmeIF4A was in the50 to 100 mg/l range. The N-terminal sequence of all preparations wereconfirmed by dire sequencing the purified protein with a Procise 494sequencer (Perkin Elmer/Applied Biosystems Division). Western blotanalyses of rLmeIF4A with a rabbit anti-sera made against the L.braziliensis eIF4A protein revealed strong specific reactivity.Hereafter, LmeIF will be referred to generically as LeIF for LeishmaniaeIF protein.

Immune responses to LmeIF by L. major infected BALB/c mice. Infection ofBALB/c mice with L. major is commonly used as a model system forcell-mediated immune regulation. These mice are widely accepted asdeveloping a predominant Th2 profile by 7 to 10 days following infectionthereby resulting in disease progression. Female, BALB/cByJ andBALB/cByJmn-scid/J mice mice were from Jackson Laboratory (Bar Harbour,Me.) and were age-matched (4-6 weeks) within each of the followingexperiments.

BALB/c female mice (three per group) were immunized with recombinant 8E(a L. braziliensis antigen unrelated to LbeIF4A) or LmeIF in PBS. Eachmouse received 70 μg of the indicated antigen in a final volume of 200μl administered subcutaneously and distributed over three sites on theshaved flank. Inguinal, brachial, axillary, and periaortic lymph nodeswere removed on day 10.

For proliferation, the lymph node cells were cultured for 72 hours at adensity of 4×10⁵/well in 96-well flat bottom plates in the presence ofvarious concentrations of the indicated antigen. The cultures werepulsed with ³[H]-thymidine for the final 16 hours of culture and wereharvested onto filters. The incorporation of radioactivity wasdetermined using a Matrix 96 Direct Beta Counter (Packard InstrumentCo., Inc).

For infection studies, L. major (Friedlan strain) promastigotes werecultured at 26° C. in M199 (GibcoBRL, Gaithersburg, Md.) containing 10%FCS (Hyclone, Logan, Utah). For in vitro responses, BALB/c mice wereinfected with 2×10⁵ stationary phase L. major promastigotes in each hindfootpad. At 10 and 28 days post-infection, animals were sacrificed andpopliteal lymph nodes removed. Single cell suspensions were preparedfrom the nodes, plated at density of either 2×10⁵ cells per well (96well flat bottom plates) for analysis of proliferative responses or at2×10⁶ cells per well (24 well plate) for cytokine analyses. Cells werepulsed with 1 μCi of [³H]-thymidine after 72 hours of culture andincorporation of radioactivity was determined approximately 16 hourslater. Levels of cytokines (IFN-γ and IL-4) secreted into the culturesupernatants after 72 hours of culture were measured by ELISA.

The levels of IFN-γ, IL-10, and IL-4 were determined by sandwich ELISA,using antibody pairs and procedures available from PharMingen. Alldeterminations of cytokine levels were derived by testing serialdilutions of the supernatants. Standard curves were generated usingrecombinant mouse cytokines available from Immunex (IL-4) or Genzyme(IL-10 and IFN-γ). The ELISAs for IFN-γ and IL-10 were sensitive to 100pg/ml of the appropriate cytokine and the ELISA for IL-4 was sensitiveto 20 pg/ml. Hamster anti-CD3 (500A2, gift of Dr. J. P. Allison, U. C.Berkeley, Berkeley, Calif.) was purified at Immunex. Since IL-10production always correlated with IL-4 production, but IL-4 productiondid not always correlate with IL-10 production, the results for the Thprofiles are shown in terms of IL-4 vs IFN-γ production only.

Results. Culture supernatants of antigen-stimulated lymph node cellswere analyzed for the production IL-4 and IFN-γ. When cells frominfected mice were stimulated in vitro with SLA. high levels of IL-4 andvery little IFN-γ were detected. However, the same cultures whenstimulated with rLmeIF produced high levels of IFN-γ and no IL-4. Thesame result was obtained using mice at both 10 (FIG. 24A) and 28 (FIG.24B) days after infection, by which time a clear Th2 pattern establishedin terms of disease progression. rLmeIF also elicited strongproliferative responses from draining lymph node cells of these L.major-infected BALB/c mice at both early (10 days) and late days) stagesinfection. By comparison, rLmSTI1 (a recently described immunogenic L.major antigen; Webb et al., J. Immunol. 157:5034-5041,1996) yielded amixed cytokine profile.

To complement the cytokine data, anti-LeIF antibody titers in sera fromBALB/c mice at 28 days post infection were analyzed for anti-LeIFantibodies (FIG. 24C). rLeIF was diluted in coating buffer (15 mMNaHCO₃t28 mM NaH₂CO₃, pH 9.6) and plated onto Coming Easy Wash ELISAplates (Corning Glass Works, Corning, N.Y.) at 1 μg/wells and incubatedovernight at 4° C. Plates were then blocked at room temperature with PBScontaining 1% BSA for 1 hour). BALB/c mice were infected with 2×10⁵stationary phase L. major promastigotes in each hind footpad and wereused as source of infection sera at 8 weeks post-infection. 100 μl ofsera diluted in PBS containing 0.1% BSA and 0.1% Tween-20 were added andincubated at room temperature for 30 minutes. Following removal ofunbound antibodies (five washes with PBS containing 0.1% Tween-20),bound antibodies were detected with goat anti-mouse IgG horseradishperoxidase-conjugated secondary antibody (Southern BiotechnologyAssociates Inc., Birmingham, Ala.). Plates were developed using TMB andread a t 500 nm. Only very low levels of anti-LeIF antibody weredetected at a serum dilution of 1:1,000. In contrast, the same micedevelop extremely high LmSTI1-specific titers (>1:200,000) and highantibody titers to SLA. This demonstrates that LeIF induces relativelyweak B cell responses during L. major infection.

rLeIF down regulates the in vitro production of SLA induced IL-4 fromdraining lymph nodes of infected Balb/c mice. To evaluate the ability ofrLeIF to down regulate the production of IL-4 by lymphocytes of L. majorinfected mice, mice were infected for 28 days with L. major, followed byculturing of lymph node cells with SLA. This resulted in the productionof IL4, but not IFN-γ by the lymph cells. The addition of variousconcentrations of rLeIF and a fixed amount of SLA to the lymph nodecultures caused an early complete abrogation of the SLA-induced IL-4secretion in a dose dependent manner (FIG. 25). It was also observedthat SLA had no effect on LeIF induced IFN-γ production.

These results indicate that lymph node cells from Leishmania infectedBALB/c mice that are stimulated in vitro with soluble leishmanialantigen (SLA), produce a cytokine profile biased towards IL-4. However,the same cells stimulated with LmeIF4A produced high amounts of IFN-γand no detectable IL-4. The addition of LmeIF4A to SLA resulted indecreased IL-4 production, demonstrating the ability of LmeIF4A to downregulate a Th2 response.

B. Th Responses to LmeIF4A in Naive BALB/c Mice Following ImmunizationWith LbeIF4A in the Absence of Added Adjuvant.

Because IL-12 is a key cytokine that favors the development of Th1responses, the ability of LeIF to induce a LeIF specific Th1 profile innaive BALB/c mice in the absence of added adjuvant was evaluated. Inthis set of experiments, the L. braziliensis eIF homologue (LbeIF) wasused as the immunizing antigen. Animals were primed with either rLbeIFor r8E (another recombinant leishmanial antigen which stimulates patientPBMC to produce high levels of IL-10) in PBS. Both antigens contain thesame 4 kD, β-gal N-terminal fusion sequence.

Draining lymph node cells from these animals were first cultured for tendays at 6×10⁶/well in a 2 ml volume in the presence of 0.5 μg/ml 8E or10 μg/ml LmeIF. The short term T cell lines were then cloned by limitingdilution in the presence of 8E or LmeIF (0.5 or 10 mg/ml, respectively),irradiated BALB/c splenocytes (2.5×10⁵/well) and IL-2 (10 μg/ml). Threeweeks later the resulting clones were resuspended in a total volume of300 μl and were transferred into wells containing immobilized anti-CD3(0.5 μg/well of a 48 well plate). Supernatants were collected 48 hourslater.

T cell lines were assayed for IFN-γ and IL-4. It was found that ˜90% ofthe T-cell clones isolated from LeIF-primed mice were Th1, producingIFN-γ and no IL-4, while the remaining 10% were Th0 producing both IFN-γand IL-4 (FIG. 26). In contrast, clones from mice primed with 8E had amixed cytokine profile.

These results suggest that the ability of LeIF to drive a specific Th1response is mediated by an adjuvant activity of LeIF itself.

C. Ability of LmeIF4A to Influence the Early Cytokine Profile inSplenocytes of SCID Mice.

To examine the activity of LeIF on spleen cells in the absence of Tcells, the above experiments were repeated using splenocytes from SCIDmice of two different genetic backgrounds both Leishmania resistant(C3H) and Leishmania susceptible (BALB/c). Briefly, cells from spleensof 6 week old female SCID mice were cultured at 2×10⁶ per well andstimulated with 10 μg/ml with rLeIF or SLA. At 12, 24, and 72 hours,supernatants from the induced cultures were harvested and the levels ofIFN-γ measured. The results demonstrate that LeIF can stimulate theproduction of high levels of IFN-γ by SCID mouse spleen cells in bothgenetic background (FIG. 27A). In contrast SLA did not stimulate theproduction of detectable IFN-γ. The IFN-γ production induced by LeIF inSCID splenocytes was IL-12 mediated; the addition of anti-IL-12 antibodyabrogated the production of IFN-γ in these cultures (FIG. 27 B). Theseresults demonstrate that in SCID mice, LeIF can stimulate IL-12production which would subsequently stimulate NK cells to produce IFN-γ.

Within a further experiment, cytokine mRNA expression was evaluated inSCID mice splenocytes stimulated with rLeIF. Splenocytes from C3H SCIDmice were cultured at 5×10⁶ cells/well (0.6 ml) in the absence (−) orpresence (+) of 10 μg/ml of rLeIF for 24 hours. Total RNA was isolatedand RT-PCR assays performed using cytokine specific primers as indicatedin FIG. 28. The results indicate that LeIF increases the level of IFN-γand IL-18 mRNA, but not IL-10 mRNA.

D. Synergistic Effect Between LeIF and Cytokines

A synergistic effect of LeIF and certain cytokines was identified inSCID mouse splenocytes. In one experiment, SCID mouse splenocytes(2×10⁶/ml) were stimulated with IL-18 and LeIF (10 μg/ml) for 72 hours.The supernatant was harvested and the level of IFN-γ assayed asdescribed above. The results, presented in FIG. 29, show that the levelof IFN-γ increases with increasing amounts of IL-18 in the presence ofLeIF.

In a similar experiment, SCID mouse splenocytes (2×10⁶/ml) werestimulated with IL-18 and IL-15 (10 or 100 ng/ml) in the presence orabsence of LeIF (10 μg/ml) for 72 hours. The supernatant was harvestedand the level of IFN-γ assayed as described above. The results,presented in FIGS. 30 and 31, show that the presence of IL-15 furtherenhances the LeIF-induced IFN-γ production in the presence of IL-18.

Within further experiments, IL-15 and IL-18 were shown to increase NKcell cytotoxic activity of SCID mouse splenocytes stimulated with LeIF.SCID mouse splenocytes were incubated with either IL-15 or IL-18 (100ng/ml), IL-12 (10 U/ml) or both in the presence or absence of LeIF (10μg/ml) for 24 hours. NK-cell activity was assayed by ⁵¹Cr release fromYAC-1 as a target cell. The results in FIGS. 32 and 33 shows that LeIFenhances the cytotoxic activity of the NK cells stimulated by IL-15 orIL-18.

D. Initial Mapping of Active Regions

The mapping of the active region(s) was initiated by constructing threeoverlapping LeIF deletions. The recombinant clones were designed toencode the N-terminal half (amino acid residues 1-226), the middleportion (residues 129-261) and the C-terminal half (residues 196-403) ofLeIF; (FIG. 2B). Purified proteins were subsequently evaluated for theirability to stimulate IFN-γ production splenocytes from C3H SCID mice byharvesting the cultures at 72 hours post stimulation with LeIF. As shownin FIG. 27C, the N-terminal half of LeIF retained the ability to induceIFN-γ with levels that were generally higher than observed with fulllength LeIF. The middle fragment (129-261) stimulated the production ofvery low levels of IFN-γ in the same assay. No dectectable IFN-γ wasfound in supernatants of splenocytes following stimulation with theC-terminal half of LeIF.

The results described herein indicate that LbeIF4A and LmeIF4Apolypeptides are powerful and selective activators for Th1 cytokinesthat may be used as a prophylactic and therapeutic vaccine antigen forleishmaniasis. Such polypeptides act through T cell dependent andindependent pathways to stimulate production of IL-12. In the T celldependent mechanism (as in the generation of LeIF specific Th1 clones),activated LeIF specific CD4⁺ T cells would induce IL-12 production frommacrophage through CD40-ligand CD40 interactions. However, the abilityof LeIF to stimulate splenocytes from SCID mice to produce IFN-γ is anovel finding and suggests that in this T cell independent pathway, LeIFacts by stimulating IL-12 production directly from monocyte/macrophageswhich would subsequently stimulate NK cells to produce IFN-γ. Thissurprising finding demonstrates that LeIF can, in the absence of otherleishmanial antigens, adjuvant its own T cell response and may helpexplain the reasons for a predominant bias towards the generation ofLeIF specific Th1 clones.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention.

14 1618 base pairs nucleic acid single linear CDS 115..1326 1 CCACTCTCTCGGTCGTCTGT CTCCCACGCG CGCACGCAGT TGATTTCCGC CTTCTTAAAC 60 GCTCTCTTTTTTTTTATTTT TCACCTGACC AACCGCACCA CGTCGGCCTC CATC ATG 117 Met 1 TCG CAGCAA GAC CGA GTT GCC CCA CAG GAC CAG GAC TCG TTC CTC GAC 165 Ser Gln GlnAsp Arg Val Ala Pro Gln Asp Gln Asp Ser Phe Leu Asp 5 10 15 GAC CAG CCCGGC GTC CGC CCG ATC CCG TCC TTC GAT GAC ATG CCG TTG 213 Asp Gln Pro GlyVal Arg Pro Ile Pro Ser Phe Asp Asp Met Pro Leu 20 25 30 CAC CAG AAC CTTCTG CGC GGC ATC TAC TCG TAC GGC TTC GAG AAA CCG 261 His Gln Asn Leu LeuArg Gly Ile Tyr Ser Tyr Gly Phe Glu Lys Pro 35 40 45 TCC AGC ATC CAG CAGCGC GCC ATC GCC CCC TTC ACG CGC GGC GGC GAC 309 Ser Ser Ile Gln Gln ArgAla Ile Ala Pro Phe Thr Arg Gly Gly Asp 50 55 60 65 ATC ATC GCG CAG GCGCAG TCC GGT ACC GGC AAG ACG GGC GCC TTC TCC 357 Ile Ile Ala Gln Ala GlnSer Gly Thr Gly Lys Thr Gly Ala Phe Ser 70 75 80 ATC GGC CTG CTG CAG CGCCTG GAC TTC CGC CAC AAC CTG ATC CAG GGC 405 Ile Gly Leu Leu Gln Arg LeuAsp Phe Arg His Asn Leu Ile Gln Gly 85 90 95 CTC GTG CTC TCC CCG ACC CGCGAG CTG GCC CTG CAG ACG GCG GAG GTG 453 Leu Val Leu Ser Pro Thr Arg GluLeu Ala Leu Gln Thr Ala Glu Val 100 105 110 ATC AGC CGC ATC GGC GAG TTCCTG TCG AAC AGC GCG AAG TTC TGT GAG 501 Ile Ser Arg Ile Gly Glu Phe LeuSer Asn Ser Ala Lys Phe Cys Glu 115 120 125 ACC TTT GTG GGT GGC ACG CGCGTG CAG GAT GAC CTG CGC AAG CTG CAG 549 Thr Phe Val Gly Gly Thr Arg ValGln Asp Asp Leu Arg Lys Leu Gln 130 135 140 145 GCT GGC GTC GTC GTC GCCGTG GGG ACG CCG GGC CGC GTG TCC GAC GTG 597 Ala Gly Val Val Val Ala ValGly Thr Pro Gly Arg Val Ser Asp Val 150 155 160 ATC AAG CGC GGC GCG CTGCGC ACC GAG TCC CTG CGC GTG CTG GTG CTC 645 Ile Lys Arg Gly Ala Leu ArgThr Glu Ser Leu Arg Val Leu Val Leu 165 170 175 GAC GAG GCT GAT GAG ATGCTG TCT CAG GGC TTC GCG GAT CAG ATT TAC 693 Asp Glu Ala Asp Glu Met LeuSer Gln Gly Phe Ala Asp Gln Ile Tyr 180 185 190 GAG ATC TTC CGC TTC CTGCCG AAG GAC ATC CAG GTC GCG CTC TTC TCC 741 Glu Ile Phe Arg Phe Leu ProLys Asp Ile Gln Val Ala Leu Phe Ser 195 200 205 GCC ACG ATG CCG GAG GAGGTG CTG GAG CTG ACA AAG AAG TTC ATG CGC 789 Ala Thr Met Pro Glu Glu ValLeu Glu Leu Thr Lys Lys Phe Met Arg 210 215 220 225 GAC CCC GTA CGC ATTCTC GTG AAG CGC GAG AGC CTG ACG CTG GAG GGC 837 Asp Pro Val Arg Ile LeuVal Lys Arg Glu Ser Leu Thr Leu Glu Gly 230 235 240 ATC AAG CAG TTC TTCATC GCC GTC GAG GAG GAG CAC AAG CTG GAC ACG 885 Ile Lys Gln Phe Phe IleAla Val Glu Glu Glu His Lys Leu Asp Thr 245 250 255 CTG ATG GAC CTG TACGAG ACC GTG TCC ATC GCG CAG TCC GTC ATC TTC 933 Leu Met Asp Leu Tyr GluThr Val Ser Ile Ala Gln Ser Val Ile Phe 260 265 270 GCC AAC ACC CGC CGCAAG GTG GAC TGG ATC GCC GAG AAG CTG AAT CAG 981 Ala Asn Thr Arg Arg LysVal Asp Trp Ile Ala Glu Lys Leu Asn Gln 275 280 285 AGC AAC CAC ACC GTCAGC AGC ATG CAC GCC GAG ATG CCC AAG AGC GAC 1029 Ser Asn His Thr Val SerSer Met His Ala Glu Met Pro Lys Ser Asp 290 295 300 305 CGC GAG CGC GTCATG AAC ACC TTC CGC AGC GGC AGC TCC CGC GTG CTC 1077 Arg Glu Arg Val MetAsn Thr Phe Arg Ser Gly Ser Ser Arg Val Leu 310 315 320 GTA ACG ACC GACCTC GTG GCC CGC GGC ATC GAC GTG CAC CAC GTG AAC 1125 Val Thr Thr Asp LeuVal Ala Arg Gly Ile Asp Val His His Val Asn 325 330 335 ATC GTC ATC AACTTC GAC CTG CCG ACG AAC AAG GAG AAC TAC CTG CAC 1173 Ile Val Ile Asn PheAsp Leu Pro Thr Asn Lys Glu Asn Tyr Leu His 340 345 350 CGC ATT GGC CGCGGC GGC CGC TAC GGC GTA AAG GGT GTT GCC ATC AAC 1221 Arg Ile Gly Arg GlyGly Arg Tyr Gly Val Lys Gly Val Ala Ile Asn 355 360 365 TTC GTG ACG GAGAAA GAC GTG GAG CTG CTG CAC GAG ATC GAG GGG CAC 1269 Phe Val Thr Glu LysAsp Val Glu Leu Leu His Glu Ile Glu Gly His 370 375 380 385 TAC CAC ACGCAG ATC GAT GAG CTC CCG GTG GAC TTT GCC GCC TAC CTC 1317 Tyr His Thr GlnIle Asp Glu Leu Pro Val Asp Phe Ala Ala Tyr Leu 390 395 400 GGC GAG TGAGCGGGCCCCT GCCCCCCTTC CCTGCCCCCC TCTCGCGACG 1366 Gly Glu AGAGAACGCACATCGTAACA CAGCCACGCG AACGATAGTA AGGGCGTGCG GCGGCGTTCC 1426 CCTCCTCCTGCCAGCGGCCC CCCTCCGCAG CGCTTCTCTT TTGAGAGGGG GGCAGGGGGA 1486 GGCGCTGCGCCTGGCTGGAT GTGTGCTTGA GCTTGCATTC CGTCAAGCAA GTGCTTTGTT 1546 TTAATTATGCGCGCCGTTTT GTTGCTCGTC CCTTTCGTTG GTGTTTTTTC GGCCGAAACG 1606 GCGTTTAAAGCA 1618 403 amino acids amino acid linear protein 2 Met Ser Gln Gln AspArg Val Ala Pro Gln Asp Gln Asp Ser Phe Leu 1 5 10 15 Asp Asp Gln ProGly Val Arg Pro Ile Pro Ser Phe Asp Asp Met Pro 20 25 30 Leu His Gln AsnLeu Leu Arg Gly Ile Tyr Ser Tyr Gly Phe Glu Lys 35 40 45 Pro Ser Ser IleGln Gln Arg Ala Ile Ala Pro Phe Thr Arg Gly Gly 50 55 60 Asp Ile Ile AlaGln Ala Gln Ser Gly Thr Gly Lys Thr Gly Ala Phe 65 70 75 80 Ser Ile GlyLeu Leu Gln Arg Leu Asp Phe Arg His Asn Leu Ile Gln 85 90 95 Gly Leu ValLeu Ser Pro Thr Arg Glu Leu Ala Leu Gln Thr Ala Glu 100 105 110 Val IleSer Arg Ile Gly Glu Phe Leu Ser Asn Ser Ala Lys Phe Cys 115 120 125 GluThr Phe Val Gly Gly Thr Arg Val Gln Asp Asp Leu Arg Lys Leu 130 135 140Gln Ala Gly Val Val Val Ala Val Gly Thr Pro Gly Arg Val Ser Asp 145 150155 160 Val Ile Lys Arg Gly Ala Leu Arg Thr Glu Ser Leu Arg Val Leu Val165 170 175 Leu Asp Glu Ala Asp Glu Met Leu Ser Gln Gly Phe Ala Asp GlnIle 180 185 190 Tyr Glu Ile Phe Arg Phe Leu Pro Lys Asp Ile Gln Val AlaLeu Phe 195 200 205 Ser Ala Thr Met Pro Glu Glu Val Leu Glu Leu Thr LysLys Phe Met 210 215 220 Arg Asp Pro Val Arg Ile Leu Val Lys Arg Glu SerLeu Thr Leu Glu 225 230 235 240 Gly Ile Lys Gln Phe Phe Ile Ala Val GluGlu Glu His Lys Leu Asp 245 250 255 Thr Leu Met Asp Leu Tyr Glu Thr ValSer Ile Ala Gln Ser Val Ile 260 265 270 Phe Ala Asn Thr Arg Arg Lys ValAsp Trp Ile Ala Glu Lys Leu Asn 275 280 285 Gln Ser Asn His Thr Val SerSer Met His Ala Glu Met Pro Lys Ser 290 295 300 Asp Arg Glu Arg Val MetAsn Thr Phe Arg Ser Gly Ser Ser Arg Val 305 310 315 320 Leu Val Thr ThrAsp Leu Val Ala Arg Gly Ile Asp Val His His Val 325 330 335 Asn Ile ValIle Asn Phe Asp Leu Pro Thr Asn Lys Glu Asn Tyr Leu 340 345 350 His ArgIle Gly Arg Gly Gly Arg Tyr Gly Val Lys Gly Val Ala Ile 355 360 365 AsnPhe Val Thr Glu Lys Asp Val Glu Leu Leu His Glu Ile Glu Gly 370 375 380His Tyr His Thr Gln Ile Asp Glu Leu Pro Val Asp Phe Ala Ala Tyr 385 390395 400 Leu Gly Glu 1867 base pairs nucleic acid single linear CDS117..1325 3 CTTTATTGTT GATTTCCGCC TTCTGAACAG CCCTCATTTT TTTTTGGTTTACCTCTCGTT 60 GCTTGTGACG CCCCTCCCCC TCTTCACCCA TCAAGCACCC CCTGTCGTCCTCCATC 116 ATG GCG CAG AAT GAT AAG ATC GCC CCC CAG GAC CAG GAC TCC TTCCTC 164 Met Ala Gln Asn Asp Lys Ile Ala Pro Gln Asp Gln Asp Ser Phe Leu1 5 10 15 GAT GAC CAG CCC GGC GTT CGC CCG ATC CCG TCC TTC GAC GAC ATGCCG 212 Asp Asp Gln Pro Gly Val Arg Pro Ile Pro Ser Phe Asp Asp Met Pro20 25 30 CTG CAC CAG AAC CTG CTG CGT GGC ATC TAC TCG TAC GGG TTC GAG AAG260 Leu His Gln Asn Leu Leu Arg Gly Ile Tyr Ser Tyr Gly Phe Glu Lys 3540 45 CCG TCC AGC ATC CAG CAG CGC GCG ATA GCC CCC TTC ACG CGC GGC GGC308 Pro Ser Ser Ile Gln Gln Arg Ala Ile Ala Pro Phe Thr Arg Gly Gly 5055 60 GAC ATC ATC GCG CAG GCC CAG TCC GGT ACC GGC AAG ACG GGT GCC TTC356 Asp Ile Ile Ala Gln Ala Gln Ser Gly Thr Gly Lys Thr Gly Ala Phe 6570 75 80 TCC ATC GGT CTG CTG CAG CGC CTG GAC TTC CGC CAC AAC CTG ATC CAG404 Ser Ile Gly Leu Leu Gln Arg Leu Asp Phe Arg His Asn Leu Ile Gln 8590 95 GGC CTC GTG CTC TCC CCC ACT CGC GAG CTG GCC CTG CAG ACG GCG GAG452 Gly Leu Val Leu Ser Pro Thr Arg Glu Leu Ala Leu Gln Thr Ala Glu 100105 110 GTG ATC AGC CGC ATC GGT GAG TTC CTG TCG AAC AGC TCC AAG TTC TGC500 Val Ile Ser Arg Ile Gly Glu Phe Leu Ser Asn Ser Ser Lys Phe Cys 115120 125 GAG ACC TTT GTC GGC GGC ACG CGC GTG CAG GAT GAC CTG CGC AAG CTG548 Glu Thr Phe Val Gly Gly Thr Arg Val Gln Asp Asp Leu Arg Lys Leu 130135 140 CAG GCC GGC GTC ATC GTT GCC GTG GGC ACG CCG GGC CGC GTG TCC GAC596 Gln Ala Gly Val Ile Val Ala Val Gly Thr Pro Gly Arg Val Ser Asp 145150 155 160 GTG ATC AAG CGT GGC GCG CTG CGC ACA GAG TCG CTG CGC GTG CTGGTG 644 Val Ile Lys Arg Gly Ala Leu Arg Thr Glu Ser Leu Arg Val Leu Val165 170 175 CTC GAC GAG GCT GAT GAG ATG CTG TCT CAG GGC TTC GCG GAC CAGATT 692 Leu Asp Glu Ala Asp Glu Met Leu Ser Gln Gly Phe Ala Asp Gln Ile180 185 190 TAC GAG ATC TTC CGC TTC CTG CCG AAG GAC ATC CAG GTC GCG CTCTTC 740 Tyr Glu Ile Phe Arg Phe Leu Pro Lys Asp Ile Gln Val Ala Leu Phe195 200 205 TCC GCC ACG ATG CCG GAG GAG GTA CTG GAG CTG ACG AAG AAG TTCATG 788 Ser Ala Thr Met Pro Glu Glu Val Leu Glu Leu Thr Lys Lys Phe Met210 215 220 CGC GAC CCC GTG CGT ATT CTC GTG AAG CGC GAG AGC CTG ACG CTGGAG 836 Arg Asp Pro Val Arg Ile Leu Val Lys Arg Glu Ser Leu Thr Leu Glu225 230 235 240 GGC ATC AAG CAG TTC TTC ATC GCC GTC GAA GAG GAG CAC AAGCTG GAC 884 Gly Ile Lys Gln Phe Phe Ile Ala Val Glu Glu Glu His Lys LeuAsp 245 250 255 ACG CTG ATG GAC CTG TAC GAG ACC GTG TCC ATC GCG CAG TCCGTC ATC 932 Thr Leu Met Asp Leu Tyr Glu Thr Val Ser Ile Ala Gln Ser ValIle 260 265 270 TTC GCC AAC ACG CGC CGC AAG GTG GAC TGG ATC GCC GAG AAGCTG AAC 980 Phe Ala Asn Thr Arg Arg Lys Val Asp Trp Ile Ala Glu Lys LeuAsn 275 280 285 CAG AGC AAC CAC ACC GTC AGC AGC ATG CAC GCC GAG ATG CCCAAG AGC 1028 Gln Ser Asn His Thr Val Ser Ser Met His Ala Glu Met Pro LysSer 290 295 300 GAC CGC GAG CGC GTC ATG AAC ACC TTC CGC AGC GGC AGC TCCCGC GTG 1076 Asp Arg Glu Arg Val Met Asn Thr Phe Arg Ser Gly Ser Ser ArgVal 305 310 315 320 CTC GTC ACG ACC GAC CTC GTG GCG CGC GGT ATC GAT GTGCAC CAC GTG 1124 Leu Val Thr Thr Asp Leu Val Ala Arg Gly Ile Asp Val HisHis Val 325 330 335 AAC ATC GTC ATC AAC TTC GAC CTG CCA ACG AAC AAG GAGAAC TAC CTG 1172 Asn Ile Val Ile Asn Phe Asp Leu Pro Thr Asn Lys Glu AsnTyr Leu 340 345 350 CAT CGC ATT GGT CGC GGC GGC CGC TAC GGC CGT AAG GGTGTT GCC ATC 1220 His Arg Ile Gly Arg Gly Gly Arg Tyr Gly Arg Lys Gly ValAla Ile 355 360 365 AAC TTC GTG ACG GAG AAG GAC GTG GAG CTA CTG CAC GAGATC GAG GCG 1268 Asn Phe Val Thr Glu Lys Asp Val Glu Leu Leu His Glu IleGlu Ala 370 375 380 CAC TAC CAC ACG CAG ATC GAC GAG CTC CCG GTC GAC TTCGCT GCC TAC 1316 His Tyr His Thr Gln Ile Asp Glu Leu Pro Val Asp Phe AlaAla Tyr 385 390 395 400 CTT GGC GAG TAA GCGGGTCCTT GCCTCCCCCC CCCTCCTCCTCCATCCCCAT 1368 Leu Gly Glu CCCCCACCAC CCCACACACC CCCCCCCCGT TCCTCGTCGGAAGAAGAAAG GACGCACATC 1428 GCCACGCGAA GGATGACGAG GGCTGAGGAG GAGCTCAGGGAACGGACTCG TCCCCGGTGA 1488 GCGGGGGGAG GAGGAGGTGA GGCCATCGCG CGAGCGCACCGCCGGAAGGT CGACCAGGGC 1548 GCTCAACACC CACCCAGCAC CCCGGTAGTT CCCTGCCCTCTCGTGCGCCT CCTCTCCCAC 1608 CCCGTAAATC TCCTGACGAC TTTGTGTGGA CCACACACGCGCGCTCTCGC TCCGTATCGG 1668 ACGCGCCCTA TACAACACAA CGAACCCGCC AACGTGCCGGTCGNCTTGTG GATGTGTGTC 1728 TGGCGTAGAA CGTGCGTCTG CCCCCGTCCC ATCCCCATCCCACCTCCTCG NGTGTGTGTG 1788 TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG AGTGTGTGTGTTTAAAAACT ATNATNTAGA 1848 ATATATATCT ATATAGGTN 1867 403 amino acidsamino acid linear protein 4 Met Ala Gln Asn Asp Lys Ile Ala Pro Gln AspGln Asp Ser Phe Leu 1 5 10 15 Asp Asp Gln Pro Gly Val Arg Pro Ile ProSer Phe Asp Asp Met Pro 20 25 30 Leu His Gln Asn Leu Leu Arg Gly Ile TyrSer Tyr Gly Phe Glu Lys 35 40 45 Pro Ser Ser Ile Gln Gln Arg Ala Ile AlaPro Phe Thr Arg Gly Gly 50 55 60 Asp Ile Ile Ala Gln Ala Gln Ser Gly ThrGly Lys Thr Gly Ala Phe 65 70 75 80 Ser Ile Gly Leu Leu Gln Arg Leu AspPhe Arg His Asn Leu Ile Gln 85 90 95 Gly Leu Val Leu Ser Pro Thr Arg GluLeu Ala Leu Gln Thr Ala Glu 100 105 110 Val Ile Ser Arg Ile Gly Glu PheLeu Ser Asn Ser Ser Lys Phe Cys 115 120 125 Glu Thr Phe Val Gly Gly ThrArg Val Gln Asp Asp Leu Arg Lys Leu 130 135 140 Gln Ala Gly Val Ile ValAla Val Gly Thr Pro Gly Arg Val Ser Asp 145 150 155 160 Val Ile Lys ArgGly Ala Leu Arg Thr Glu Ser Leu Arg Val Leu Val 165 170 175 Leu Asp GluAla Asp Glu Met Leu Ser Gln Gly Phe Ala Asp Gln Ile 180 185 190 Tyr GluIle Phe Arg Phe Leu Pro Lys Asp Ile Gln Val Ala Leu Phe 195 200 205 SerAla Thr Met Pro Glu Glu Val Leu Glu Leu Thr Lys Lys Phe Met 210 215 220Arg Asp Pro Val Arg Ile Leu Val Lys Arg Glu Ser Leu Thr Leu Glu 225 230235 240 Gly Ile Lys Gln Phe Phe Ile Ala Val Glu Glu Glu His Lys Leu Asp245 250 255 Thr Leu Met Asp Leu Tyr Glu Thr Val Ser Ile Ala Gln Ser ValIle 260 265 270 Phe Ala Asn Thr Arg Arg Lys Val Asp Trp Ile Ala Glu LysLeu Asn 275 280 285 Gln Ser Asn His Thr Val Ser Ser Met His Ala Glu MetPro Lys Ser 290 295 300 Asp Arg Glu Arg Val Met Asn Thr Phe Arg Ser GlySer Ser Arg Val 305 310 315 320 Leu Val Thr Thr Asp Leu Val Ala Arg GlyIle Asp Val His His Val 325 330 335 Asn Ile Val Ile Asn Phe Asp Leu ProThr Asn Lys Glu Asn Tyr Leu 340 345 350 His Arg Ile Gly Arg Gly Gly ArgTyr Gly Arg Lys Gly Val Ala Ile 355 360 365 Asn Phe Val Thr Glu Lys AspVal Glu Leu Leu His Glu Ile Glu Ala 370 375 380 His Tyr His Thr Gln IleAsp Glu Leu Pro Val Asp Phe Ala Ala Tyr 385 390 395 400 Leu Gly Glu 407amino acids amino acid <Unknown> linear 5 Met Ser Gly Gly Ser Ala AspTyr Asn Arg Glu His Gly Gly Pro Glu 1 5 10 15 Gly Met Asp Pro Asp GlyVal Ile Glu Ser Asn Trp Asn Glu Ile Val 20 25 30 Asp Asn Phe Asp Asp MetAsn Leu Lys Glu Ser Leu Leu Arg Gly Ile 35 40 45 Tyr Ala Tyr Gly Phe GluLys Pro Ser Ala Ile Gln Gln Arg Ala Ile 50 55 60 Ile Pro Cys Ile Lys GlyTyr Asp Val Ile Ala Gln Ala Gln Ser Gly 65 70 75 80 Thr Gly Lys Thr AlaThr Phe Ala Ile Ser Ile Leu Gln Gln Leu Glu 85 90 95 Ile Glu Phe Lys GluThr Gln Ala Leu Val Leu Ala Pro Thr Arg Glu 100 105 110 Leu Ala Gln GlnIle Gln Lys Val Ile Leu Ala Leu Gly Asp Tyr Met 115 120 125 Gly Ala ThrCys His Ala Cys Ile Gly Gly Thr Asn Val Arg Asn Glu 130 135 140 Met GlnLys Leu Gln Ala Glu Ala Pro His Ile Val Val Gly Thr Pro 145 150 155 160Gly Arg Val Phe Asp Met Leu Asn Arg Arg Tyr Leu Ser Pro Lys Trp 165 170175 Ile Lys Met Phe Val Leu Asp Glu Ala Asp Glu Met Leu Ser Arg Gly 180185 190 Phe Lys Asp Gln Ile Tyr Glu Arg Val Gln Lys Leu Asn Thr Ser Ile195 200 205 Gln Val Val Leu Leu Ser Ala Thr Met Pro Thr Asp Val Leu GluVal 210 215 220 Thr Lys Lys Phe Met Arg Asp Pro Ile Arg Ile Leu Val LysLys Glu 225 230 235 240 Glu Leu Thr Leu Glu Gly Ile Lys Gln Phe Tyr IleAsn Val Glu Arg 245 250 255 Glu Glu Trp Lys Leu Asp Thr Leu Cys Asp LeuTyr Glu Thr Leu Thr 260 265 270 Ile Thr Gln Ala Val Ile Phe Leu Asn ThrArg Arg Lys Val Asp Trp 275 280 285 Leu Thr Glu Lys Met Gln Ala Ile TyrPhe Thr Val Ser Ala Leu His 290 295 300 Gly Asp Met Asp Gln Lys Glu ArgAsp Val Ile Met Arg Glu Phe Arg 305 310 315 320 Ser Gly Ser Ser Arg ValLeu Ile Thr Thr Asp Leu Leu Ala Arg Gly 325 330 335 Ile Asp Val Gln GlnVal Ser Leu Val Ile Asn Tyr Asp Leu Pro Thr 340 345 350 Asn Arg Glu AsnTyr Ile His Arg Ile Gly Arg Gly Gly Arg Phe Gly 355 360 365 Arg Lys GlyVal Ala Ile Asn Phe Val Thr Glu Glu Asp Lys Arg Ile 370 375 380 Leu ArgHis Ile Glu Thr Phe Tyr Asn Thr Thr Val Glu Glu Met Pro 385 390 395 400Met Asn Gly Ala Asp Leu Ile 405 407 amino acids amino acid <Unknown>linear 6 Met Ser Gly Gly Ser Ala Asp Tyr Asn Arg Glu His Gly Gly Pro Glu1 5 10 15 Gly Met Asp Pro Asp Gly Val Ile Glu Ser Asn Trp Asn Glu IleVal 20 25 30 Asp Asn Phe Asp Asp Met Asn Leu Lys Glu Ser Leu Leu Arg GlyIle 35 40 45 Tyr Ala Tyr Gly Phe Glu Lys Pro Ser Ala Ile Gln Gln Arg AlaIle 50 55 60 Ile Pro Cys Ile Lys Gly Tyr Asp Val Ile Ala Gln Ala Gln SerGly 65 70 75 80 Thr Gly Lys Thr Ala Thr Phe Ala Ile Ser Ile Leu Gln GlnLeu Glu 85 90 95 Ile Glu Phe Lys Glu Thr Gln Ala Leu Val Leu Ala Pro ThrArg Glu 100 105 110 Leu Ala Gln Gln Ile Gln Lys Val Ile Leu Ala Leu GlyAsp Tyr Met 115 120 125 Gly Ala Thr Cys His Ala Cys Ile Gly Gly Thr AsnVal Arg Asn Glu 130 135 140 Met Gln Lys Leu Gln Ala Glu Ala Pro His IleVal Val Gly Thr Pro 145 150 155 160 Gly Arg Val Phe Asp Met Leu Asn ArgArg Tyr Leu Ser Pro Lys Trp 165 170 175 Ile Lys Met Phe Val Leu Asp GluAla Asp Glu Met Leu Ser Arg Gly 180 185 190 Phe Lys Asp Gln Ile Tyr GluIle Phe Gln Lys Leu Asn Thr Ser Ile 195 200 205 Gln Val Val Phe Ala SerAla Thr Met Pro Thr Asp Val Leu Glu Val 210 215 220 Thr Lys Lys Phe MetArg Asp Pro Ile Arg Ile Leu Val Lys Lys Glu 225 230 235 240 Glu Leu ThrLeu Glu Gly Ile Lys Gln Phe Tyr Ile Asn Val Glu Arg 245 250 255 Glu GluTrp Lys Leu Asp Thr Leu Cys Asp Leu Tyr Glu Thr Leu Thr 260 265 270 IleThr Gln Ala Val Ile Phe Leu Asn Thr Arg Arg Lys Val Asp Trp 275 280 285Leu Thr Glu Lys Met His Ala Arg Asp Phe Thr Val Ser Ala Leu His 290 295300 Gly Asp Met Asp Gln Lys Glu Arg Asp Val Ile Met Arg Glu Phe Arg 305310 315 320 Ser Gly Ser Ser Arg Val Leu Ile Thr Thr Asp Leu Leu Ala ArgGly 325 330 335 Ile Asp Val Gln Gln Val Ser Leu Val Ile Asn Tyr Asp LeuPro Thr 340 345 350 Asn Arg Glu Asn Tyr Ile His Arg Ile Gly Arg Gly GlyArg Phe Gly 355 360 365 Arg Lys Gly Val Ala Ile Asn Phe Val Thr Glu GluAsp Lys Arg Ile 370 375 380 Leu Arg Asp Ile Glu Thr Phe Tyr Asn Thr ThrVal Glu Glu Met Pro 385 390 395 400 Met Asn Val Ala Asp Leu Ile 405 54base pairs nucleic acid single linear 7 CAATTACATA TGCATCACCA TCACCATCACATGGCGCAGA ATGATAAGAT CGCC 54 34 base pairs nucleic acid single linear 8CATGGAATTC CGCTTACTCG CCAAGGTAGG CAGC 34 37 base pairs nucleic acidsingle linear 9 CATGGAATTC TTAGTCGCGC ATGAACTTCT TCGTCAG 37 54 basepairs nucleic acid single linear 10 CAATTACATA TGCATCACCA TCACCATCACTTCCGCTTCC TGCCGAAGGA CATC 54 54 base pairs nucleic acid single linear11 CAATTACATA TGCATCACCA TCACCATCAC GAGACCTTTG TCGGCGGCAC GCGC 54 37base pairs nucleic acid single linear 12 CATGGAATTC TTACAGGTCCATCAGCGTGT CCAGCTT 37 4 amino acids amino acid <Unknown> linear 13 AspGlu Ala Asp 1 5 amino acids amino acid <Unknown> linear 14 Gly Thr GlyLys Thr 1 5

I claim:
 1. An isolated polypeptide comprising SEQ ID NO:
 4. 2. Anisolated polypeptide comprising amino acids 49-403 of SEQ ID NO:
 4. 3.An isolated polypeptide comprising a portion of SEQ ID NO: 4, whereinthe portion possesses the ability to stimulate a Th1 response inperipheral blood mononuclear cells.
 4. A composition comprising apolypeptide according to any one of claims 1, 2 or 3 and aphysiologically acceptable carrier.